Liquid pharmaceutical composition

ABSTRACT

The present invention relates to novel liquid pharmaceutical compositions of adalimumab, which include adalimumab or a biosimilar thereof, an acetate buffering agent/system such as sodium acetate/acetic acid, and a sugar stabiliser such as trehalose. Such a combination of components furnishes formulations having a stability (e.g. on storage and when exposed to stress) which is comparable to or an improvement upon those known in the art, and with fewer ingredients. Such advances will help adalimumab treatments to become more widely available at lower cost, and prolong the viability of pre-loaded delivery devices (e.g. pre-filled syringes) to reduce unnecessary waste of the drug.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/313,470, issued as U.S. Pat. No. 10,426,832 on Oct. 1, 2019, which isa U.S. national stage application under 35 U.S.C. § 371 of InternationalPatent Application No. PCT/EP2015/060816, which claims priority to andthe benefit of European Patent Application No. 14169753.2, filed May 23,2014, the contents of each of which are incorporated by reference hereinin their entireties.

INTRODUCTION

The present invention relates to a novel protein formulation. Inparticular, the invention relates to a liquid pharmaceutical compositionof adalimumab, to a method of manufacturing the composition, to a kitincluding the composition, to a package including the composition, to amethod of manufacturing the package, and to methods of treatment usingthe composition and/or package.

BACKGROUND

Treatment of tumour necrosis factor-alpha (TNF-α)-related autoimmunediseases, such as rheumatoid arthritis, psoriasis and other autoimmunediseases, has been achieved through the use of FDA-approved drugs suchas Adalimumab (HUMIRA®, Abbott Corporation). Adalimumab is a humanmonoclonal antibody that inhibits human TNF-α activity so as to preventit from activating TNF receptors, thereby downregulating inflammatoryresponses associated with autoimmune diseases. Approved medicalindications for Adalimumab include rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,moderate to severe chronic psoriasis and juvenile idiopathic arthritis.

Adalimumab is generally delivered to a patient via subcutaneousinjection, and is thus provided in a liquid form, typically in packagessuch as vials, preloaded syringes, or preloaded “pen devices”.Commercially available pen devices (HUMIRA® Pen) generally include a 1mL pre-filled glass syringe, preloaded with 0.8 mL of a sterileformulation of 40 mg Adalimumab (see below), with a fixed needle (eithergray natural rubber or a latex free version) and a needle cover.Commercial formulations (HUMIRA®) of Adalimumab contain the followingingredients:

Amount per container (mg) Ingredient (filling volume = 0.8 mL) Amount(mg/mL) Adalimumab 40 50 Citric Acid 1.04 1.3 Monohydrate Dibasicsodium1.22 1.53 phosphate dihydrate Mannitol 9.6 12 Monobasicsodium 0.69 0.86phosphate dihydrate Polysorbate 80 0.8 1 Sodium chloride 4.93 6.16Sodium citrate 0.24 0.3 WFI and sodium q.b. to adjust pH to 5.2 q.b. toadjust pH to 5.2 hydroxide

Adalimumab, and its method of manufacture, is described in WO97/29131(BASF) as D2E7, and elsewhere in the art.

Though the aforementioned commercial formulation of Adalimumab is stable(at least to some extent), the relevant antibody may be unstable overprolonged periods or under stressed conditions, thus precludingprolonged storage of said formulations. Such degradation of theformulation may be due to a variety of factors, including:

-   -   Physical effects, such as:        -   Inadequate inhibition of aggregation of the relevant protein            molecules (a function supposedly served by Tween®80            (polysorbate 80));        -   Inadequate inhibition of precipitation;        -   Inadequate inhibition of adsorption of the relevant protein            molecules at the interface of water and air or at the            contact surface of any packaging material (a function            supposedly served by Tween®80 (polysorbate 80));        -   Inadequate regulation of osmotic pressure (a function            supposedly served by mannitol);    -   Chemical effects, such as:        -   Inadequate regulation of oxidation (a function supposedly            served by mannitol and potentially undermined by Tween®80            (polysorbate 80), which can promoted oxidation of double            bonds);        -   Inadequate inhibition of photo-oxidation;        -   Inadequate inhibition of hydrolysis of ester bonds leading            to the formation of acid, aldehyde and peroxide products,            thus affecting the stability of the antibody;        -   Inadequate stabilisation and maintenance of pH;        -   Inadequate inhibition of protein fragmentation;        -   Inadequate inhibition of protein unfolding;

Any, some, or all of the above factors can lead to either an unviabledrug product (which may be unsafe for use in medical treatments) or adrug product whose viability is variable and unpredictable, especiallyin view of the variable stresses (agitation, heat, light) differentbatches of drug product may be exposed to during manufacture, transport,and storage.

In terms of the physical and chemical stabilisation of Adalimumab, thecomplex array of components within the aforementioned commercialformulations appears to perform below expectations, especially in viewof the large number of components. Though this particular combination ofexcipients undoubtably represents a ‘delicate balance’ (given theinterplay between various technical factors) and was the result ofextensive research and development, in view of the risk ofunderperformance it is questionable whether such a large number ofdifferent excipients is justified, especially given that this inevitablyincreases processing and cost burdens, toxicity risks, and risks ofdeleterious interactions between components that could compromise theformulation. Even if the overall performance of the commercialformulations could not be surpassed, an alternative formulation havingcomparative performance but containing few components would represent ahighly desirable replacement for the commercial formulations, for atleast the aforesaid reasons.

In order to guarantee reproducible clinical performance of aprotein-based pharmaceutical product, such products must remain in astable and consistent form over time. It is well-established thatmolecular alterations can occur during every stage of the manufacturingprocess, including during the production of the final formulation andduring storage. Molecular alterations can modify a quality attribute ofa biopharmaceutical product, resulting in an undesirable change in theidentity, strength or purity of the product. Some such problems areoutlined above.

The primary goal of formulation development is to provide apharmaceutical composition that will support the stability of abiopharmaceutical protein during all stages of its production, storage,shipping and use. Formulation development for an innovativebiopharmaceutical protein, or a biosimilar monoclonal antibody (mAb), isessential to its safety, clinical efficacy and commercial success.

There is therefore a need for the provision of alternative or improvedliquid formulations of adalimumab. Desirably, any new formulations wouldsolve at least one of the aforementioned problems and/or at least oneproblem inherent in the prior art, and may suitably solve two or more ofsaid problems. Desirably, the problem(s) of the prior art may be solvedwhilst reducing the complexity of the formulation.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention there is provided aliquid pharmaceutical composition comprising adalimumab (which suitablyincludes any biosimilar thereof); an acetate buffering agent (or anacetate buffer system); and a sugar stabiliser; wherein the compositionoptionally comprises (or excludes) any one or more additional componentsdefined herein in relation to a liquid pharmaceutical composition (e.g.including surfactant, excluding arginine, etc.), optionally in anyamount, concentration, or form stipulated herein; and wherein thecomposition optionally exhibits any one or more parameters or propertiesgiven herein in relation to a liquid pharmaceutical composition (e.g.pH, osmolality, aggregation, fragmentation, protein unfolding,turbidity, etc.).

According to a second aspect of the present invention there is provideda liquid pharmaceutical composition comprising adalimumab; an acetatebuffering agent (or an acetate buffer system); and a sugar stabiliser;wherein the composition is either (substantially or entirely) free ofarginine (suitably L-arginine) or comprises arginine in a concentrationof at most 0.1 mM.

According to a third aspect of the present invention there is provided aliquid pharmaceutical composition comprising adalimumab; an acetatebuffering agent (or an acetate buffer system); and a sugar stabiliser;wherein the composition is either (substantially or entirely) free ofarginine or comprises arginine in a molar ratio of arginine to acetatebuffering agent of at most 1:150 (i.e. less than or equal to one mole ofarginine for every 150 moles of acetate buffering agent).

According to a fourth aspect of the present invention there is provideda liquid pharmaceutical composition comprising adalimumab; an acetatebuffering agent (or an acetate buffer system); and a sugar stabiliser;wherein the composition is either (substantially or entirely) free ofarginine or comprises arginine in a weight ratio of arginine toadalimumab of at most 1:3000 (i.e. less than or equal to one part byweight of arginine for every 3000 parts by weight adalimumab).

According to a fifth aspect of the present invention there is provided apackage (e.g. pre-filled syringe, pen, intravenous bag, or apackage/container containing any of the aforementioned) comprising aliquid pharmaceutical composition as defined herein.

According to a sixth aspect of the present invention there is provided adrug delivery device (e.g. pre-filled syringe or pen, or intravenousbag) comprising a liquid pharmaceutical composition as defined herein.

According to a seventh aspect of the present invention there is provideda kit of parts comprising a drug delivery device, a liquidpharmaceutical composition as defined herein (optionally contained in apackage or container), and optionally a set of instructions withdirections regarding the administration (e.g. sub-cutaneous) of theliquid pharmaceutical composition.

According to an eighth aspect of the present invention there is provideda method of manufacturing a liquid pharmaceutical composition, themethod comprising mixing together adalimumab; an acetate buffering agent(or an acetate buffer system); a sugar stabiliser; and optionally anyone or more additional components defined herein in relation to a liquidpharmaceutical composition, optionally in any amount, concentration, orform stipulated; and optionally adjusting any one or more parametersgiven herein in relation to a liquid pharmaceutical composition (e.g.pH, osmolality).

According to a ninth aspect of the present invention there is provided aliquid pharmaceutical composition obtainable by, obtained by, ordirectly obtained by a method of manufacturing a liquid pharmaceuticalcomposition as defined herein.

According to a tenth aspect of the present invention there is provided amethod of manufacturing a package or a drug delivery device, the methodcomprising incorporating a liquid pharmaceutical composition as definedherein within a package or drug delivery device.

According to an eleventh aspect of the present invention there isprovided a package or a drug delivery device obtainable by, obtained by,or directly obtained by a method of manufacturing a package or a drugdelivery device as defined herein.

According to a twelfth aspect of the present invention there is provideda method of treating a disease or medical disorder in a patient in needof such treatment, said method comprising administering to said patienta therapeutically effective amount of a liquid pharmaceuticalcomposition as defined herein.

According to a thirteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein therapy.

According to a fourteenth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of a disease ordisorder.

According to a fifteenth aspect of the present invention there isprovided a method of treating a tumour necrosis factor-alpha(TNF-α)-related autoimmune disease in a patient in need of suchtreatment, said method comprising administering to said patient atherapeutically effective amount of a liquid pharmaceutical compositionas defined herein.

According to a sixteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein the treatment of a tumour necrosis factor-alpha (TNF-α)-relatedautoimmune disease.

According to a seventeenth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of a tumournecrosis factor-alpha (TNF-α)-related autoimmune disease.

According to an eighteenth aspect of the present invention there isprovided a method of treating rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis in apatient in need of such treatment, said method comprising administeringto said patient a therapeutically effective amount of a liquidpharmaceutical composition as defined herein.

According to a nineteenth aspect of the present invention there isprovided a liquid pharmaceutical composition as defined herein for usein the treatment of rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis.

According to a twentieth aspect of the present invention there isprovided a use of a liquid pharmaceutical composition as defined hereinin the manufacture of a medicament for the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis.

In further aspects, the invention provides a liquid pharmaceuticalcomposition, a package, a drug delivery device, a kit of parts, a methodof manufacturing a liquid pharmaceutical composition, a method ofmanufacturing a package or a drug delivery device, a method of treating,a liquid pharmaceutical composition for use, and a use of a liquidpharmaceutical composition in the manufacture of a medicament,essentially as defined herein (including in any of the aforementionedtwenty aspects) except that, rather than being specific to “adalimumab”(and biosimilars thereof), the invention may instead apply (and therebybe defined as relating) to any TNF-α-inhibiting antibody (anti-TNF-αantibody) (or any biosimilar thereof), albeit suitably an antibody thatinhibits human TNF-α activity, and most suitably a human monoclonalantibody that inhibits human TNF-α activity. Suitably the anti-TNF-αantibody is a therapeutically effective medicament (at least whenadministered in appropriate quantities to a patient in need thereof) (ora biosimilar thereof—see below for definitions of biosimilars inrelation to adalimumab, which applies equally to all anti-TNF-αantibodies), suitably one which has received FDA approval. As such, anyreference herein to “adalimumab” may, unless incompatible therewith, beconstrued as a reference to any anti-TNF-α antibody for the purpose ofthese additional aspects of the invention (whether this relates toabsolute or relative amounts, concentrations, parameters, or properties,or whether it relates to certain definitions, such as what constitutes abiosimilar).

One of these further aspects of the present invention provides a liquidpharmaceutical composition comprising an anti-TNF-α antibody (whichsuitably includes any biosimilar thereof); an acetate buffering agent(or an acetate buffer system); and a sugar stabiliser; wherein thecomposition optionally comprises (or excludes) any one or moreadditional components defined herein in relation to a liquidpharmaceutical composition (e.g. including surfactant, excludingarginine, etc.), optionally in any amount, concentration, or formstipulated herein; and wherein the composition optionally exhibits anyone or more parameters or properties given herein in relation to aliquid pharmaceutical composition (e.g. pH, osmolality, aggregation,fragmentation, protein unfolding, turbidity, etc.).

In a particular embodiment, the anti-TNF-α antibody is selected from thegroup including adalimumab, infliximab, certolizumab pegol, golimumab.

Any features, including optional, suitable, and preferred features,described in relation to any particular aspect of the invention may alsobe features, including optional, suitable and preferred features, of anyother aspect of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, and to show how embodimentsof the same are put into effect, reference is now made, by way ofexample, to the following diagrammatic drawings, in which:

FIG. 1 is a bar chart showing the protein content (mg/mL), as determinedby OD, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after 4 weeks (red bars)of the formulation(s) being heated at 40° C.

FIG. 2 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (orange bars) of the formulation(s) being heated at40° C.

FIG. 3 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (dark blue bars, time=0) and after both 2 weeks(pink bars) and 4 weeks (light blue bars) of the formulation(s) beingheated at 40° C.

FIG. 4 is a bar chart showing the unfolding temperature (° C.), asdetermined by DSF, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations).

FIG. 5 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (red bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (purple bars) of the formulation(s) being heated at40° C.

FIG. 6 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (blue bars, time=0) and after both 2 weeks (redbars) and 4 weeks (green bars) of the formulation(s) being heated at 40°C.

FIG. 7 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)at an arbitrary start point (blue bars, time=0) and after both 2 weeks(red bars) and 4 weeks (green bars) of the formulation(s) being heatedat 40° C.

FIG. 8 is a bar chart showing the acid cluster peak(s) isoforms profile,as determined by iCE280 analysis, of the DoE2 formulations (of Example2) at an arbitrary start point (blue bars, time=0) and after both 2weeks (red bars) and 4 weeks (green bars) of the formulation(s) beingheated at 40° C.

FIG. 9 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 2 weeks (red bars) and 4weeks (green bars) of the formulation(s) being heated at 40° C.

FIG. 10 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2) at an arbitrary startpoint (blue bars, time=0) and after both 24 hours (red bars) and 48hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 11 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 12 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

FIG. 13 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), beforeexposure to light (blue bars, time=0) and after 7-hour light exposure at765 W/m² (red bars).

FIG. 14 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars).

FIG. 15 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2),along with reference standards (representing comparator HUMIRA®formulations), before exposure to light (blue bars, time=0) and after7-hour light exposure at 765 W/m² (red bars).

FIG. 16 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2), along with reference standards (representing comparatorHUMIRA® formulations), before exposure to light (blue bars, time=0) andafter 7-hour light exposure at 765 W/m² (red bars).

FIG. 17 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars).

FIG. 18 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)before (blue bars, time=0) and after m² (red bars) five freeze-thawingcycles (−80° C.→room temperature).

FIG. 19 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2) before (blue bars, time=0) and after m² (red bars) fivefreeze-thawing cycles (−80° C.→room temperature).

FIG. 20 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations) before (bluebars, time=0) and after m² (red bars) five freeze-thawing cycles (−80°C.→room temperature).

FIG. 21 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 10microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

FIG. 22 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 25microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specificationand claims have the following meanings set out below.

References herein to “adalimumab” include the originator drug substance(as commercially available), adalimumab as defined in WO97/29131 (BASF)(particularly D2E7 therein) and elsewhere in the art, and alsobiosimilars thereof. D2E7 of WO97/29131 “has a light chain CDR3 domaincomprising the amino acid sequence of SEQ ID NO: 3 and a heavy chainCDR3 domain comprising the amino acid sequence of SEQ ID NO: 4”.Preferably, the D2E7 antibody has a light chain variable region (LCVR)comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chainvariable region (HCVR) comprising the amino acid sequence of SEQ ID NO:2. WO97/29131 gives details of each of these sequence listings.References herein to “adalimumab” may include biosimilars which, forinstance, may share at least 75%, suitably at least 80%, suitably atleast 85%, suitably at least 90%, suitably at least 95%, suitably atleast 96%, suitably at least 97%, suitably at least 98% or most suitablyat least 99% protein sequence identity with any one of protein sequencesdisclosed in either WO97/29131 (especially in relation to D2E7) orelsewhere in relation to “adalimumab”. Alternatively or additionally,references herein to “adalimumab” may include biosimilars which exhibitat least 75%, suitably at least 80%, suitably at least 85%, suitably atleast 90%, suitably at least 95%, suitably at least 96%, suitably atleast 97%, suitably at least 98% or most suitably at least 99% proteinsequence homology with any one of protein sequences disclosed in eitherWO97/29131 (especially in relation to D2E7) or elsewhere in relation to“adalimumab”. Alternatively or additionally, a biosimilar may have a(slightly) different glycosylation profile, even if the protein sequenceis substantially the same or different to the extent specified above.

The term “biosimilar” (also known as follow-on biologics) is well knownin the art, and the skilled person would readily appreciate when a drugsubstance would be considered a biosimilar of adalimumab. Furthermore,such “biosimilars” would need to be officially approved as a“biosimilar” for marketing before said “biosimilar” is sold on the openmarket. The term “biosimilar” is generally used to describe subsequentversions (generally from a different source) of “innovatorbiopharmaceutical products” (“biologics” whose drug substance is made bya living organism or derived from a living organism or throughrecombinant DNA or controlled gene expression methodologies) that havebeen previously officially granted marketing authorisation. Sincebiologics have a high degree of molecular complexity, and are generallysensitive to changes in manufacturing processes (e.g. if different celllines are used in their production), and since subsequent follow-onmanufacturers generally do not have access to the originator's molecularclone, cell bank, know-how regarding the fermentation and purificationprocess, nor to the active drug substance itself (only the innovator'scommercialized drug product), any “biosimilar” is unlikely to be exactlythe same as the innovator drug product.

For the purposes of various molar calculations (e.g. for molar ratiosbetween adalimumab and another component of the liquid pharmaceuticalcomposition of the invention) the molecular weight of adalimumab may betaken to be 144190.3 g/mol (reference molecular weight) based on detailsdisclosed on the CAS database for CAS #331731-18-1, Adalimumab, wherethe molecular formula is taken as C₆₄₂₈H₉₉₁₂N₁₆₉₄O₁₉₈₇S₄₆. As such, aliquid pharmaceutical composition containing 50 mg/mL adalimumab may beconsidered a 0.347 mM (or 347 μM) solution of adalimumab. This is notintended to be in any way limiting regarding the nature of anybiosimilars of adalimumab covered by the scope of the present invention,nor the level of glycosylation, either of which may affect the actualmolecular weight. However, where a biosimilar does have a differentmolecular weight, the abovementioned reference molecular weight shouldbe suitably used for the purposes of assessing whether or not such abiosimilar falls within the scope of any molar definitions stipulatedwithin this specification. So the number of moles in a known weight ofsaid biosimilar should be calculated, just for the purposes of thisinvention, using the above reference molecular weight.

Herein, the term “buffer” or “buffer solution” refers to a generallyaqueous solution comprising a mixture of an acid (usually a weak acid,e.g. acetic acid, citric acid, imidazolium form of histidine) and itsconjugate base (e.g. an acetate or citrate salt, for example, sodiumacetate, sodium citrate, or histidine) or alternatively a mixture of abase (usually a weak base, e.g. histidine) and its conjugate acid (e.g.protonated histidine salt). The pH of a “buffer solution” will changevery only slightly upon addition of a small quantity of strong acid orbase due to the “buffering effect” imparted by the “buffering agent”.

Herein, a “buffer system” comprises one or more buffering agent(s)and/or an acid/base conjugate(s) thereof, and more suitably comprisesone or more buffering agent(s) and an acid/base conjugate(s) thereof,and most suitably comprises one buffering agent only and an acid/baseconjugate thereof. Unless stated otherwise, any concentrationsstipulated herein in relation to a “buffer system” (i.e. a bufferconcentration) suitably refers to the combined concentration of thebuffering agent(s) and/or acid/base conjugate(s) thereof. In otherwords, concentrations stipulated herein in relation to a “buffer system”suitably refer to the combined concentration of all the relevantbuffering species (i.e. the species in dynamic equilibrium with oneanother, e.g. acetate/acetic acid). As such, a given concentration of anacetate buffer system generally relates to the combined concentration ofacetate (or acetate salt(s), e.g. sodium acetate) and acetic acid. Theoverall pH of the composition comprising the relevant buffer system isgenerally a reflection of the equilibrium concentration of each of therelevant buffering species (i.e. the balance of buffering agent(s) toacid/base conjugate(s) thereof).

Herein, the term “buffering agent” refers to an acid or base component(usually a weak acid or weak base) of a buffer or buffer solution. Abuffering agent helps maintain the pH of a given solution at or near toa pre-determined value, and the buffering agents are generally chosen tocomplement the pre-determined value. A buffering agent is suitably asingle compound which gives rise to a desired buffering effect,especially when said buffering agent is mixed with (and suitably capableof proton exchange with) an appropriate amount (depending on thepre-determined pH desired) of its corresponding “acid/base conjugate”,or if the required amount of its corresponding “acid/base conjugate” isformed in situ—this may be achieved by adding strong acid or base untilthe required pH is reached. By way of example:

An acetate “buffering agent” is suitably an acetate salt, for example,sodium acetate, suitably mixed with its acid/base conjugate, aceticacid. Such a buffer system may be formed by simply mixing a given amountof sodium acetate with a given amount of acetic acid. Alternatively,however, such a buffer may be formed by adding a given amount of a base,suitably a strong base (e.g. sodium hydroxide) to the acetic acid untilthe desired pH (and thus the desired balance of sodium acetate/aceticacid) is reached. Herein, except where the contrary is stated, anyconcentrations given in relation to an acetate buffer or acetatebuffering agent suitably refer to the combined concentration of thebuffering agent(s) (e.g. sodium acetate) and/or acid/base conjugate(s)thereof (e.g. acetic acid). The skilled person is readily able tocalculate such concentrations. Such concentrations may be calculated byreference to the combined concentrations of buffering agent(s) andacid/base conjugate(s), where a buffer system is formed by simply mixingtogether buffering agent(s) and acid/base conjugate(s). Alternatively,where a buffer system is formed by mixing either the buffering agent(s)or acid/base conjugate(s) with a pH adjuster (e.g. strong acid or strongbase) to produce a mixture of each, suitably such concentrations may becalculated by reference to the starting amounts/concentrations of thebuffering agent(s) or acid/base conjugate(s) respectively. For example,where a buffer system is formed using a known amount/concentration ofacetic acid which is mixed with a pH adjuster (e.g. sodium hydroxide)until the desired pH is reached, the concentration of the buffer systemmay be calculated by reference to the initial amount of acetic acid.

Herein, an “acid/base conjugate” refers to the conjugate acid orconjugate base (whichever is relevant at a particular pH—typically theconjugate acid in the context of the present invention) of a particular“buffering agent”. The acid/base conjugate of an acetate buffering agent(e.g. sodium acetate) is suitably acetic acid.

Herein, the term “buffering species” refers to the particular species(excluding any associated counteranions or countercations—i.e. ignoresodium ions for sodium acetate/acetic acid systems) of a given buffersystem which are in dynamic equilibrium with (and proton-exchange with)one another. For example, acetate anions and acetic acid togetherconstitute the “acetate buffering species” of a “acetate buffer system”.

Since it is somewhat difficult to define quantities (whether absolute orrelative) of a buffer system by reference to weight (since the totalweight will depend on the desired pH, which will affect the amount ofcounterions present), herein weight-based quantities may instead bedetermined by reference to a theoretical weight of the relevant“buffering species”. At least two species are present in any given setof “buffering species” (in relative amounts that can only be determinedby reference to the pH), each with a different molecular weight (whichusually differs by just 1). Therefore, to enable viable weightcalculations and references, for the purposes of this specification theweight of any given set of “buffering species” is given as a theoreticalweight based on just one of the buffering species, namely the mostacidic of the buffering species (i.e. the most protonated form at anygiven pH). So the weight of a given set of “buffering species” is quotedas the weight of acid-species equivalents. By way of example, in anacetate buffer system the acetate buffering species may consist ofacetate anions (ignore countercations) and acetic acid. The weight ofthe “buffering species” is therefore calculated as if acetic acid wasthe only species present in the buffer system (even though acetate isclearly present alongside acetic acid). Thus, any reference to a weightor weight ratio involving a “acetate buffering species” suitably refersto the theoretical weight of acetic acid equivalents within the buffersystem. As such, where a composition is formed by adding a pH adjuster(e.g. sodium hydroxide) to a fixed amount of acetic acid, the originalweight of acetic acid may be considered to be the weight of the“buffering species” regardless of the ultimate pH. Alternatively, if theconcentration (i.e. molarity) of a buffer system is known, this can beconverted into a weight of “buffering species” by reference to themolecular weight of the most acidic form of the relevant bufferingspecies (e.g. acetic acid), and ignoring the fact that acetate anionsare also present.

Unless stated otherwise, references herein to an “amino acid” or “aminoacids”, whether specific (e.g. arginine, histidine) or general (e.g. anyamino acid), in the context of their presence or otherwise withincompositions (especially pharmaceutical liquid compositions of theinvention) relate to the corresponding free amino acid(s) (regardless ofits/their protonation state and/or salt form, though for consistencyamounts are suitably calculated by reference to the free amino acid perse). This may suitably include natural and/or artificial amino acids.Unless stated to the contrary, such references are not intended torelate to amino acid residue(s) covalently incorporated as part of alarger compound (as opposed to a composition comprising multiplecompounds), such as a peptide or protein (where such amino acid residuesare linked via peptide bonds). As such, though adalimumab, as a protein,contains amino acid residues, it is not considered to comprise any “freeamino acid(s)”. By way of example, a composition defined as being “freeof arginine” does not contain any free arginine but it may still includeone or more proteins (e.g. adalimumab) which do themselves comprisearginine residues.

Unless stated otherwise, references herein to any one or more “aminoacids”, whether specific or general, suitably relate to theL-stereoisomers or a racemate thereof, most suitably L-amino acids.

The term “substantially free”, when used in relation to a givencomponent of a composition (e.g. “a liquid pharmaceutical compositionsubstantially free of arginine”), refers to a composition to whichessentially none of said component has been added. As explained above,such references have no bearing on the presence of amino acid residue(s)within a protein structure. When a composition is “substantially free”of a given component, said composition suitably comprises no more than0.001 wt % of said component, suitably no more than 0.0001 wt % of saidcomponent, suitably no more than 0.00001 wt %, suitably no more than0.000001 wt %, suitably no more than 0.0000001 wt % thereof, mostsuitably no more than 0.0001 parts per billion (by weight).

The term “entirely free”, when used in relation to a given component ofa composition (e.g. “a liquid pharmaceutical composition substantiallyfree of arginine”), refers to a composition containing none of saidcomponent. As explained above, such references have no bearing on thepresence of amino acid residue(s) within a protein structure.

Herein, in the context of the present specification, a “strong acid” issuitably one having a pK_(a) of −1.0 or less, whereas a “weak acid” issuitably one having a pK_(a) of 2.0 or more. Herein, in the context ofthe present specification, a “strong base” is suitably one whoseconjugate acid has a pK_(a) of 12 or higher (suitably 14 or higher),whereas a “weak base” is suitably one whose conjugate acid has a pK_(a)of 10 or less.

Herein, a “stabiliser” refers to a component which facilitatesmaintenance of the structural integrity of the biopharmaceutical drug,particularly during freezing and/or lyophilization and/or storage(especially when exposed to stress). This stabilising effect may arisefor a variety of reasons, though typically such stabilisers may act asosmolytes which mitigate against protein denaturation. Typicalstabilisers include amino acids (i.e. free amino acids not part of apeptide or protein—e.g. glycine, arginine, histidine, aspartic acid,lysine) and sugar stabilisers, such as a sugar polyol (e.g. mannitol,sorbitol), and/or a disaccharide (e.g. trehalose, sucrose, maltose,lactose), though the liquid pharmaceutical compositions of the inventioninclude a stabiliser, at least one of which is a sugar stabiliser (i.e.either a sugar polyol or a disaccharide). Most suitably the at least onesugar stabiliser is a non-reducing sugar (be it a sugar polyol or adisaccharide).

Herein, a “non-reducing sugar” is generally a sugar without any aldehydemoieties or without the capability of forming an aldehyde moiety (e.g.through isomerism).

Herein, a “tonicity modifier” or “tonicifier” refers to a reagent whoseinclusion within a composition suitably contributes to (or increases)the overall osmolality and osmolarity of the composition. Suitably, atonicifier, as used herein includes an agent which functions to render asolution similar in osmotic characteristics to physiologic fluids.

Herein, references to specific amounts of a given component of acomposition, especially a buffering agent, stabiliser, amino acid,surfactant, or tonicifier, suitably relate to the amounts of the pureanhydrous form of the relevant component (or compositions formed byusing said amounts of the pure anhydrous form), even though such acomponent may be used in a non-anhydrous form when forming thecomposition. Amounts of any corresponding non-anhydrous forms (e.g.monohydrates, dihydrates, etc.) may be readily calculated by simplyusing the appropriate multiplier. For instance, unless stated otherwise(as per the Examples, where quantities relate to trehalose dihydrate),amounts stipulated in relation to trehalose refer to the anhydrous formof trehalose (or compositions formed by using the stipulatedamounts/concentrations of anhydrous trehalose), which has a molecularweight of 342.296 g/mol, so to calculate the corresponding amount oftrehalose dihydrate needed to form the same composition (less waterwould have to be added) it is necessary to multiply the stipulatedamount by 378.33/342.296, since 378.33 is the molecular weight oftrehalose dihydrate. The skilled person would readily understand how tojudiciously adjust the quantity of diluent/water depending on the formof the components used, in order to derive the target concentrations.

Herein, the term “pharmaceutical composition” refers to a formulation ofa pharmaceutical active which renders the biological activity of theactive ingredient therapeutically effective, but which does not includeother ingredients which are obviously toxic to a subject to which theformulation are intended to be administered.

Herein, the term “stable” generally refers to the physical stabilityand/or chemical stability and/or biological stability of a component,typically an active or composition thereof, during preservation/storage.

It is to be appreciated that references to “treating” or “treatment”include prophylaxis as well as the alleviation of established symptomsof a condition. “Treating” or “treatment” of a state, disorder orcondition therefore includes: (1) preventing or delaying the appearanceof clinical symptoms of the state, disorder or condition developing in ahuman that may be afflicted with or predisposed to the state, disorderor condition but does not yet experience or display clinical orsubclinical symptoms of the state, disorder or condition, (2) inhibitingthe state, disorder or condition, i.e., arresting, reducing or delayingthe development of the disease or a relapse thereof (in case ofmaintenance treatment) or at least one clinical or subclinical symptomthereof, or (3) relieving or attenuating the disease, i.e., causingregression of the state, disorder or condition or at least one of itsclinical or subclinical symptoms.

In the context of the present invention, a “therapeutically effectiveamount” or “effective amount” of the antibody means an amount that iseffective, when administered to a mammal for treating a disease ordisorder, in prophylactic and therapeutic aspect and the antibody iseffective in treatment of the diseases concerned.

The “therapeutically effective amount” will vary depending on thecompound, the disease and its severity and the age, weight, etc., of themammal to be treated.

The term “human TNF-α” refers to the human cytokine which exists in a 17kD secreted form and a 26 kD membrane-associated form, and in abiologically active form, TNF-α could be observed as a trimer ofcovalently-bound 17 kD molecule. Its specific structure can be found inPennica, D. et al. (1984) Nature 312: 724-729; Davis, J. M. et al.(1987) Biochemistry 26, 1322-1326; and Jones, E. Y. et al. (1989) Nature338: 225-228.

The term “recombinant human antibody” is intended to include a humanantibody prepared, expressed, produced or isolated using a recombinantmethod.

Herein, amounts stipulated for components and ingredients, whetherspecified in terms of “parts”, ppm (parts per million), percentages (%,e.g. wt %), or ratios, are intended to be by weight, unless statedotherwise.

Where the quantity or concentration of a particular component of a givencomposition is specified as a weight percentage (wt % or % w/w), saidweight percentage refers to the percentage of said component by weightrelative to the total weight of the composition as a whole. It will beunderstood by those skilled in the art that the sum of weightpercentages of all components of a composition (whether or notspecified) will total 100 wt %. However, where not all components arelisted (e.g. where compositions are said to “comprise” one or moreparticular components), the weight percentage balance may optionally bemade up to 100 wt % by unspecified ingredients (e.g. a diluent, such aswater, or other non-essentially but suitable additives).

Herein, unless stated otherwise, the term “parts” (e.g. parts by weight,pbw) when used in relation to multiple ingredients/components, refers torelative ratios between said multiple ingredients/components. Expressingmolar or weight ratios of two, three or more components gives rise tothe same effect (e.g. a molar ratio of x, y, and z is x₁:y₁:z₁respectively, or a range x₁-x₂:y₁-y₂:z₁-z₂). Though in many embodimentsthe amounts of individual components within a composition may be givenas a “wt %” value, in alternative embodiments any or all such wt %values may be converted to parts by weight (or relative ratios) todefine a multi-component composition. This is so because the relativeratios between components is often more important than the absoluteconcentrations thereof in the liquid pharmaceutical compositions of theinvention. Where a composition comprising multiple ingredients isdescribed in terms of parts by weight alone (i.e. to indicate onlyrelative ratios of ingredients), it is not necessary to stipulate theabsolute amounts or concentrations of said ingredients (whether in totoor individually) because the advantages of the invention can stem fromthe relative ratios of the respective ingredients rather than theirabsolute quantities or concentrations. However, in certain embodiments,such compositions consists essentially of or consist of the stipulatedingredients and a diluents (e.g. water).

Where a composition is said to comprise a plurality of stipulatedingredients (optionally in stipulated amounts of concentrations), saidcomposition may optionally include additional ingredients other thanthose stipulated. However, in certain embodiments, a composition said tocomprise a plurality of stipulated ingredients may in fact consistessentially of or consist of all the stipulated ingredients.

Herein, where a composition is said to “consists essentially of” aparticular component, said composition suitably comprises at least 70 wt% of said component, suitably at least 90 wt % thereof, suitably atleast 95 wt % thereof, most suitably at least 99 wt % thereof. Suitably,a composition said to “consist essentially of” a particular componentconsists of said component save for one or more trace impurities.

Herein, the term “particle size” or “pore size” refers respectively tothe length of the longest dimension of a given particle or pore. Bothsizes may be measured using a laser particle size analyser and/orelectron microscopes (e.g. tunneling electron microscope, TEM, orscanning electron microscope, SEM). The particle count (for any givensize) can be obtained using the protocols and equipment outlined in theExamples, which relates to the particle count of sub-visible particles.

Liquid Pharmaceutical Composition

The present invention provides a liquid pharmaceutical composition,suitably as defined herein. The composition suitably comprises a humanmonoclonal antibody, suitably one which inhibits human TNF-α activity,suitably so as to prevent it from activating TNF receptors. Mostsuitably the liquid pharmaceutical composition comprises adalimumab,which in itself suitably includes any biosimilar thereof. Thecomposition suitably comprises an acetate buffering agent (or a acetatebuffer system). The composition suitably comprises a sugar stabiliser.The composition is suitably (substantially or entirely) free of arginineor comprises arginine either in a concentration of at most 0.1 mM, in amolar ratio of arginine to acetate buffering agent (or a acetate buffersystem) of at most 1:150, or in a weight ratio of arginine to adalimumabof at most 1:3000 (i.e. less than or equal to one part by weight ofarginine for every 3000 parts by weight acetate buffering agent).Alternatively or in addition, the composition may suitably include anyone or more additional components defined herein in relation to a liquidpharmaceutical composition (e.g. including surfactant, excludingarginine, etc.), optionally in any amount, concentration, or formstipulated herein; and wherein the composition optionally exhibits anyone or more parameters or properties given herein in relation to aliquid pharmaceutical composition (e.g. pH, osmolality).

Advantageously, the present invention provides alternative and improvedliquid pharmaceutical compositions, which generally exhibit betterstability and viability than those of the prior art. As is illustratedherein (see Examples), the liquid pharmaceutical formulations of thepresent invention have comparable or improved characteristics whencompared to the conventional formulations of adalimumab, for example thecommercially available formulation Humira®, when subjected to differentstressing conditions (thermal, mechanical and light). Their performanceis also generally comparable or better than many other comparativeformulations that were subjected to the same stress testing. Since thesestressing conditions are highly representative of the kind of stresssuch formulations are subjected to during manufacture, transport, andstorage, they provide an excellent indication of the advantages of theinvention. That such good stability performance can be achieved usingless complex formulations with fewer excipients was consideredsurprising in view of the general teachings of the prior art.

Adalimumab

Adalimumab, which is commercially available in HUMIRA® formulations, andits method of manufacture, is described in WO97/29131 (BASF) as D2E7,and elsewhere in the art. It is described as having “a light chain CDR3domain comprising the amino acid sequence of SEQ ID NO: 3 and a heavychain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 4”(WO97/29131). Furthermore, the D2E7 antibody is described as having alight chain variable region (LCVR) comprising the amino acid sequence ofSEQ ID NO: 1 and a heavy chain variable region (HCVR) comprising theamino acid sequence of SEQ ID NO: 2 (WO97/29131).

The medical indications and function of Adalimumab, are elucidatedhereinbefore.

In the context of the invention “adalimumab” includes biosimilars, asdefined herein before, and the skilled person would readily appreciatethe scope of the term “adalimumab” in the context of the invention.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab at a concentration of from about 5 to about 150 mg/ml,suitably from about 25 to about 75 mg/mL. For example, the adalimumabmay be present in the formulation at a concentration of about 25, about30, about 35, about 40, about 45, about 50, about 55, about 60, about65, about 70 or about 75 mg/ml. In an embodiment, the adalimumab ispresent at a concentration from about 45 to about 55 mg/ml. In anembodiment, the adalimumab is present at a concentration of about 50mg/ml.

Buffer, Buffering Agent, and pH

Suitably, the liquid pharmaceutical composition is a buffered solutionwhose pH is stabilised by a buffering agent (or a buffer system),suitably in combination with an acid/base conjugate of the bufferingagent. As such, the liquid pharmaceutical composition suitably comprisesa buffering agent as defined herein. Preferably, the liquidpharmaceutical composition additionally comprises an acid/baseconjugate, wherein said acid/base conjugate corresponds to the conjugateacid or conjugate base of the buffering agent, depending on whether thebuffering agent is itself a base or acid respectively. Collectively, thebuffering agent and its acid/base conjugate may be considered a “buffersystem”. The liquid pharmaceutical composition thus suitably comprises a“buffer system” (suitably comprising a buffering agent(s) and anacid/base conjugate(s) thereof), and any concentrations stipulated inrelation to the buffer system generally relate to the combinedconcentrations of the buffering agent(s) and any acid/base conjugate(s)thereof. Any “buffer system” suitably comprises a weak acid and a weakbase (see above definitions).

Suitably, the buffering agent is an acetate buffering agent. Suitablythe acetate buffering agent is an acetate salt, suitably comprisinganionic acetate (i.e. AcO⁻) and one or more pharmaceutically acceptablecountercations. A suitable acetate salt may include a metal acetate salt(e.g. an alkali metal acetate or an alkaline earth metal acetate), or anon-metal acetate salt (e.g. ammonium acetate, triethylammoniumacetate). In a particular embodiment, the buffering agent (and theacetate salt) is sodium acetate.

Suitably, the liquid pharmaceutical composition comprises an acid/baseconjugate of the buffering agent, most suitably acetic acid as theconjugate acid of an acetate salt. The combination of the bufferingagent and its acid/base conjugate constitute a buffer system. Suitably,the liquid pharmaceutical composition comprises the buffering agent andits corresponding acid/base conjugate, suitably such that together thebuffering agent and its acid/base conjugate are present at a level (i.e.absolute amount or concentration) and in a relative amount (orconcentration) sufficient to provide the desired pH for the composition.The buffer system may be formed by simply mixing the buffering agentwith its acid/base conjugate or may alternatively be formed by mixing anacid or base with either the buffering agent or its acid/base conjugatein order to form in situ the desired mixture of buffering agent andacid/base conjugate. For example, the buffer system may be formed bysimply mixing the acetate buffering agent (e.g. sodium acetate) with itsacid/base conjugate (i.e. acetic acid), suitably in a ratio appropriateto furnish the desired pH. Alternatively, the buffer system may beformed by adding a base (e.g. sodium hydroxide) to the acid/baseconjugate (i.e. acetic acid) of the acetate buffering agent, suitably inan amount appropriate to furnish the desired pH and mixture of thebuffering agent (e.g. sodium acetate) and corresponding acid/baseconjugate (i.e. acetic acid). Alternatively, either method of formingthe buffer system may be employed, and pH may be judiciously adjusted byeither adding further acid (suitably strong acid, such as HCl) orfurther base (suitably strong base, such as sodium hydroxide).

Most suitably, the buffer system is an acetate buffer system, suitablycomprising an acetate salt and acetic acid.

Suitably, the liquid pharmaceutical composition comprises at most onebuffering agent. Suitably, the liquid pharmaceutical compositioncomprises at most one buffer system.

Suitably, the liquid pharmaceutical composition has a pH greater than orequal to 5.0. Suitably, the liquid pharmaceutical composition has a pHless than or equal to 6.7.

In a particular embodiment, especially where the buffering agent is anacetate buffering agent, the liquid pharmaceutical composition has a pHbetween 5.0 and 5.5. In a particular embodiment, the liquidpharmaceutical composition has a pH between 5.1 and 5.3. In a particularembodiment, the liquid pharmaceutical composition has a pH of about 5.2.

Suitably, the liquid pharmaceutical composition comprises a buffersystem (suitably an acetate buffer system comprising an acetatebuffering agent) at a concentration of from about 2 to about 50 mM. Inan embodiment, the buffer system is present at a concentration ofbetween 5 and 14 mM, most suitably about 10 mM. In an embodiment, thebuffer system is present at a concentration of 10 mM. In an embodiment,the liquid pharmaceutical composition comprises a sodium acetate/aceticacid buffer system at a concentration of 10 mM. This includes where the“buffering agent(s)” (e.g. sodium acetate) is formed by the addition ofa strong base (e.g. sodium hydroxide) to the conjugate acid of thebuffering agent(s) (e.g. acetic acid).

Suitably, the liquid pharmaceutical composition comprises the bufferingspecies (suitably acetate buffering species) at a concentration of fromabout 0.120 mg/mL to about 3.0 mg/mL. In an embodiment, the bufferingspecies are present at a concentration of between 0.30 mg/mL and 0.84mg/mL, most suitably about 0.60 mg/mL. This includes where the“buffering agent” (e.g. sodium acetate) is formed by the addition of astrong base (e.g. sodium hydroxide) to the conjugate acid of thebuffering agent (e.g. acetic acid).

Suitably, the liquid pharmaceutical composition comprises the buffersystem (suitably the acetate buffer system) in a molar ratio of buffersystem to adalimumab of from about 5:1 to about 145:1. In an embodiment,the buffer system is present in a molar ratio of buffer system toadalimumab of from about 14:1 to about 40:1, most suitably about 29:1.In an embodiment, the buffer system is present at a concentration of29:1. This includes where the “buffering agent(s)” (e.g. sodium acetate)is formed by the addition of a strong base (e.g. sodium hydroxide) tothe conjugate acid of the buffering agent (e.g. acetic acid).

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including an acetate buffer system performparticularly well in stress tests, especially in relation tofragmentation and protein unfolding, which can be important indicatorsof stability and drug product viability. Furthermore, liquidpharmaceutical compositions whose acetate buffer system maintains asteady pH 5.2 perform particularly well.

Sugar Stabiliser

Suitably, the liquid pharmaceutical composition comprises a stabiliser,most suitably a sugar stabiliser. Suitably, such a component facilitatesmaintenance of the structural integrity of the biopharmaceutical drug,particularly during freezing and/or lyophilization and/or storage(especially when exposed to stress).

The liquid pharmaceutical composition may comprise one or more sugarstabilisers, though in preferred embodiments only a single sugarstabiliser is present.

Suitably, the sugar stabiliser is a sugar polyol (including sugaralcohols) and/or a disaccharide.

The sugar stabiliser is suitably selected from the group includingtrehalose, mannitol, sucrose, sorbitol, maltose, lactose, xylitol,arabitol, erythritol, lactitol, maltitol, inositol.

In a particular embodiment, the sugar stabiliser is selected from thegroup including trehalose, mannitol, sucrose, maltose, lactose, xylitol,arabitol, erythritol, lactitol, maltitol, inositol.

In a particular embodiment, the sugar stabiliser is a non-reducingsugar, optionally a non-reducing sugar listed anywhere herein.

In a particular embodiment, the sugar stabiliser is selected from thegroup including trehalose and mannitol.

In a particular embodiment, the sugar stabiliser is trehalose. Trehaloseis a particularly advantageous sugar stabiliser for use alongside anacetate buffering agent/buffer system in liquid adalimumab formulations.

Suitably, the liquid pharmaceutical composition comprises at most onesugar stabiliser, suitably at most one sugar polyol and/or disaccharide.Suitably, the liquid pharmaceutical composition comprises trehalose asthe only sugar stabiliser.

Suitably the trehalose used to form the liquid pharmaceuticalcomposition is trehalose dihydrate, though suitably any amountsstipulated in relation to trehalose (unless stated otherwise—as done inthe Examples) pertain to pure, anhydrous trehalose. Such amounts may beconverted into an amount of trehalose dihydrate by applying anappropriate multiplier. Moreover, for the purposes of assessing whethera given formulation falls within the scope of any of the trehalosequantity definitions given herein, an amount of trehalose dihydrate canbe readily converted into a corresponding amount of pure, anhydroustrehalose (with an equal number of moles) through applying saidmultiplier in reverse. This principle may be adopted for any sugarstabiliser component. Concentrations, when given as a molarconcentration, will of course be the same regardless of the hydrationstate of the sugar stabiliser.

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) at a concentration of from about50 to about 400 mM, more suitably from about 100 to about 300 mM, moresuitably from about 150 to about 250 mM. In an embodiment, the sugarstabilizer(s) is present at a concentration of between 190 and 210 mM,most suitably about 200 mM. In an embodiment, trehalose is present at aconcentration of 200 mM.

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) at a concentration of from about15 mg/mL to about 140 mg/mL, more suitably from about 35 mg/mL to about100 mg/mL, more suitably from about 45 mg/mL to about 80 mg/mL. In anembodiment, the sugar stabilizer(s) is present at a concentration ofbetween 65 mg/mL and 72 mg/mL, most suitably about 68 mg/mL. In aparticular embodiment, trehalose is present at a concentration of about68 mg/mL (which equates to about 75.7 mg/mL trehalose dihydrate).

Suitably, the liquid pharmaceutical composition comprises the sugarstabilizer(s) (most suitably trehalose) in a molar ratio of sugarstabilizer(s) to adalimumab of from about 145:1 to about 1150:1, moresuitably from about 290:1 to about 860:1, more suitably from about 430:1to about 720:1. In an embodiment, the sugar stabilizer(s) is present ata molar ratio of sugar stabilizer(s) to adalimumab of from about 550:1to about 605:1, most suitably about 576:1. In an embodiment, trehaloseis present at a molar ratio of trehalose to adalimumab of about 576:1.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including a sugar stabiliser as definedherein perform particularly well in stress tests, especially in relationto aggregation, fragmentation and protein unfolding, which can beimportant indicators of stability and drug product viability.Furthermore, liquid pharmaceutical compositions comprising trehalose asthe sugar stabiliser perform particularly well.

Diluent

The liquid pharmaceutical compositions of the invention may include anyone or more pharmaceutically acceptable diluents, or mixture thereof.However, most suitably the liquid pharmaceutical composition is anaqueous pharmaceutical composition. Most suitably the diluent is water,and suitably water alone. The water is suitably water for injection(WFI).

Suitably the diluent may constitute the balance of ingredients in anyliquid pharmaceutical composition, for instance so that the weightpercentages total 100%. Suitably any concentrations given herein inrelation to any component of the liquid pharmaceutical compositionrepresent concentrations of said component in (and suitably dissolvedin) the diluent in admixture with any other components.

The liquid pharmaceutical composition of the invention is suitably asolution, and is suitably (substantially or entirely) free ofparticulates or precipitates.

Absent or Low Level Components

Low/No Arginine

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine (suitably L-arginine) or comprisesarginine in a concentration of at most 0.1 mM, more suitably at most0.01 mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a molar ratio ofarginine to buffer system of at most 1:150 (i.e. less than or equal toone mole of arginine for every 150 moles of buffer system), moresuitably at most 1:1500, most suitably at most 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a weight ratio ofarginine to adalimumab of at most 1:3000 (i.e. less than or equal to onepart by weight of arginine for every 3000 parts by weight adalimumab),more suitably at most 1:30,000, most suitably at most 1:300,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of arginine or comprises arginine in a molar ratio ofarginine to adalimumab of at most 1:3.75 (i.e. less than or equal to onemole of arginine for every 3.75 moles adalimumab), more suitably at most1:37.5, most suitably at most 1:375.

As explained herein, such references to “arginine” in the context oftheir presence or otherwise within liquid pharmaceutical compositionsrelate to the corresponding free amino acid(s) and not amino acidresidue(s) covalently incorporated as part of a larger compound, such asa peptide or protein.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludearginine perform particularly well in stress tests, especially inrelation to aggregation, fragmentation and protein unfolding.

Low/No Amino Acids

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids or comprises one or more amino acids ina (collective) concentration of at most 0.1 mM, more suitably at most0.01 mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids or comprises one or more amino acids ina (collective) molar ratio of amino acids(s) to buffer system of at most1:150 (i.e. less than or equal to one mole of amino acids(s) for every150 moles of buffer system), more suitably at most 1:1500, most suitablyat most 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids or comprises one or more amino acids ina (collective) weight ratio of amino acids(s) to adalimumab of at most1:3000 (i.e. less than or equal to one part by weight of amino acids(s)for every 3000 parts by weight adalimumab), more suitably at most1:30,000, most suitably at most 1:300,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of amino acids or comprises one or more amino acids ina (collective) molar ratio of amino acid(s) to adalimumab of at most1:3.75 (i.e. less than or equal to one mole of amino acid(s) for every3.75 moles adalimumab), more suitably at most 1:37.5, most suitably atmost 1:375.

As explained herein, such references to “amino acids” in the context oftheir presence or otherwise within liquid pharmaceutical compositionsrelate to the corresponding free amino acid(s) and not amino acidresidue(s) covalently incorporated as part of a larger compound, such asa peptide or protein.

Suitably, the amino acids referred to in this section (and deemed eitherabsent or present in low quantities) may be natural and/or artificialamino acids, though they are preferably natural amino acids. Inparticular, the liquid pharmaceutical compositions are either(substantially or entirely) free of any amino acids selected from thegroup including: arginine, lysine, aspartic acid, and histidine; orcomprises one or more of the aforesaid amino acids in an amount,concentration, molar ratio, or weight ratio as hereinbefore defined inrelation to “amino acid(s)”.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludeamino acids or certain amino acids, as defined above, performparticularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding.

Low/No Surfactants

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)concentration of at most 1 mM, more suitably at most 0.1 mM, moresuitably at most 0.01 mM, more suitably at most 0.001 mM, most suitablyat most 0.0001 mM. The liquid pharmaceutical composition may, under suchcircumstances, optionally comprise polysorbate 80 as defined herein.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)molar ratio of surfactant(s) to buffer system of at most 1:10, moresuitably at most 1:100, most suitably at most 1:1000, more suitably atmost 1:10,000, suitably at most 1:100,000. The liquid pharmaceuticalcomposition may, under such circumstances, optionally comprisepolysorbate 80 as defined herein.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)weight ratio of surfactant(s) to adalimumab of at most 1:50 (i.e. lessthan or equal to one part by weight of surfactant(s) for every 50 partsby weight adalimumab), more suitably at most 1:500, more suitably atmost 1:5000, more suitably at most 1:50,000, suitably at most 1:500,000.The liquid pharmaceutical composition may, under such circumstances,optionally comprise polysorbate 80 as defined herein.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of surfactants (whether cationic, anionic, amphoteric,or non-ionic) with the optional exception of polysorbate 80(polyoxyethylene (20) sorbitan monooleate) or comprises one or more ofsaid surfactants (optionally excluding polysorbate 80) in a (collective)molar ratio of surfactant(s) to adalimumab of at most 3:1, more suitablyat most 0.3:1, more suitably 0.003:1, more suitably 0.0003:1, suitably0.00003:1. The liquid pharmaceutical composition may, under suchcircumstances, optionally comprise polysorbate 80 as defined herein.

Suitably, the surfactants referred to in this section (and deemed eitherabsent or present in low quantities) may be cationic, anionic,amphoteric, or non-ionic surfactants. Suitably, the surfactants referredto in this section (and deemed either absent or present in lowquantities) include cationic, anionic, and amphoteric surfactants, butmay optionally exclude non-ionic surfactants (e.g. polysorbates orspans) or at least may optionally exclude polysorbate 80. As such, theliquid pharmaceutical composition is either (substantially or entirely)free of cationic, anionic, or amphoteric surfactants or comprises one ormore of said surfactants in an amount, concentration, molar ratio, orweight ratio of at most that stipulated in any of the precedingparagraphs of this sub-section in relation to “surfactant(s)” moregenerally.

The liquid pharmaceutical composition is either (substantially orentirely) free of non-ionic surfactants with the optional exception ofpolysorbate 80 or comprises one or more of said surfactants in anamount, concentration, molar ratio, or weight ratio of at most thatstipulated in any of the preceding paragraphs of this sub-section inrelation to “surfactant(s)” more generally.

The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate surfactants with the optional exception ofpolysorbate 80 or comprises one or more of said surfactants in anamount, concentration, molar ratio, or weight ratio of at most thatstipulated in any of the preceding paragraphs of this sub-section inrelation to “surfactant(s)” more generally. The liquid pharmaceuticalcomposition may, under such circumstances, optionally comprisepolysorbate 80 as defined herein.

The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate 20 (also known as Tween®20—polyoxyethylene(20) sorbitan monolaurate) surfactants or comprises one or more of saidsurfactants in an amount, concentration, molar ratio, or weight ratio ofat most that stipulated in any of the preceding paragraphs of thissub-section in relation to “surfactant(s)” more generally.

The liquid pharmaceutical composition may suitably be either(substantially or entirely) free of polysorbate 80 surfactants orcomprises said surfactant(s) in an amount, concentration, molar ratio,or weight ratio as hereinbefore defined in relation to “surfactant(s)”.The liquid pharmaceutical composition is either (substantially orentirely) free of polysorbate 80 surfactants or comprises one or more ofsaid surfactants in an amount, concentration, molar ratio, or weightratio of at most that stipulated in any of the preceding paragraphs ofthis sub-section in relation to “surfactant(s)” more generally.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludesurfactants or certain surfactants, as defined above, performparticularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding.

Low/No Phosphate

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents (e.g. sodium dihydrogenphosphate, disodium hydrogen phosphate) or comprises a phosphate buffersystem in a concentration of at most 0.1 mM, more suitably at most 0.01mM, most suitably at most 0.001 mM.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents (e.g. sodium dihydrogenphosphate, disodium hydrogen phosphate) or comprises a phosphate buffersystem in a molar ratio of phosphate buffer system to any non-phosphatebuffer systems present of at most 1:150 (i.e. less than or equal to onemole of phosphate buffering agent for every 150 moles of non-phosphatebuffer system present), more suitably at most 1:1500, most suitably atmost 1:15,000.

Suitably the liquid pharmaceutical composition is either (substantiallyor entirely) free of phosphate buffering agents or comprises a phosphatebuffer system in a molar ratio of phosphate buffer system to adalimumabof at most 1:3.75 (i.e. less than or equal to one mole of phosphatebuffer system for every 3.75 moles adalimumab), more suitably at most1:37.5, most suitably at most 1:375.

References to “phosphate buffering agents” in the context of theirpresence or otherwise within liquid pharmaceutical compositions relateto any phosphate salts in any form or protonation state, includingphosphate, monohydrogen phosphate, and dihydrogen phosphate. It does,however, suitably exclude any phosphate moieties or residues that may becovalently incorporated as part of a larger compound, such as aphosphorylated or glycosylated peptide or protein.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention which (substantially or entirely) excludephosphate buffering agents perform particularly well in stress tests,especially in relation to aggregation, fragmentation and proteinunfolding.

Optional Additional Components

Tonicifier

The liquid pharmaceutical composition of the invention suitablycomprises a “tonicity modifier” (or “tonicifier”) or one or moretonicifiers, suitably as defined herein.

The inclusion of a tonicifier suitably contributes to (or increases) theoverall osmolality and osmolarity of the composition. Suitably atonicifier is present within the composition in a quantity orconcentration sufficient for the composition to be (substantially)isotonic with body fluids. Suitably a tonicifier is present within thecomposition in a quantity or concentration sufficient for thecomposition to have an osmolarity or osmolality within a range definedherein.

Any suitable tonicifier may be used. However, suitably the tonicifier isselected from the group including water-soluble metal salts (e.g. sodiumchloride, potassium chloride, magnesium chloride, calcium chloride),water-soluble tonicifying sugars/sugar alcohols (e.g. glucose, sucrose,mannitol), and/or other water-soluble polyols. Suitably thetonicifier(s) is non-buffering (i.e. gives rise to little or nobuffering effect). As such, any metal salt tonicifiers are suitably notbuffering agents.

The liquid pharmaceutical composition may comprise one or moretonicifiers, though preferably only a single “tonicifier” as such ispresent (notwithstanding any tonicifying effects imparted to thecomposition by components intended to serve another function as definedherein).

Most preferably, the tonicifier is or comprises a metal salt (preferablya non-buffering water-soluble metal salt). Suitably, said metal salt isor comprises a metal halide, suitably an alkali or an alkaline earthmetal halide, suitably an alkali metal chloride.

In a particular embodiment, the tonicifier is or comprises sodiumchloride. In a particular embodiment, the tonicifier is sodium chloride.Sodium chloride is a particularly advantageous stabiliser for usealongside an acetate buffering agent/buffer system in liquid adalimumabformulations.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) at a concentration of fromabout 10 to about 200 mM, more suitably from about 20 to about 100 mM,more suitably from about 25 to about 75 mM. In an embodiment, thetonicifier(s) is present at a concentration of between 40 and 60 mM,most suitably about 50 mM. In an embodiment, sodium chloride is presentat a concentration of 50 mM.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) at a concentration of fromabout 0.5 mg/mL to about 12 mg/mL, more suitably from about 1.2 mg/mL toabout 5 mg/mL, more suitably from about 1.5 mg/mL to about 4.4 mg/mL. Inan embodiment, the tonicifier(s) is present at a concentration ofbetween 2.7 mg/mL and 3.1 mg/mL, most suitably about 2.9 mg/mL. In aparticular embodiment, sodium chloride is present at a concentration ofabout 2.9 mg/mL.

Suitably, the liquid pharmaceutical composition comprises thetonicifier(s) (most suitably sodium chloride) in a molar ratio oftonicifier to adalimumab of from about 30:1 to about 580:1, moresuitably from about 60:1 to about 290:1, more suitably from about 70:1to about 220:1. In an embodiment, the tonicifier(s) is present at amolar ratio of tonicifier to adalimumab of from about 115:1 to about175:1, most suitably about 145:1. In an embodiment, sodium chloride ispresent at a molar ratio of sodium chloride to adalimumab of about145:1.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including a tonicifier as defined hereinperform particularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding, which can be importantindicators of stability and drug product viability. Furthermore, liquidpharmaceutical compositions comprising sodium chloride, particularly inan amount range as stipulated, perform particularly well.

Surfactant

The liquid pharmaceutical composition of the invention suitablycomprises a surfactant or one or more surfactants, suitably as definedherein.

The inclusion of a surfactant suitably contributes to stabilisation ofthe adalimumab protein.

Any suitable surfactant may be used. However, suitably the surfactant isa non-ionic surfactant, most suitably a polysorbate (polyoxyethyleneglycol sorbitan alkyl esters) or span (sorbitan alkyl esters)surfactant.

Though one or more surfactants may be included within the liquidpharmaceutical composition of the invention, most suitably only a singlesurfactant is present, most suitably a single non-ionic surfactant(suitably as defined herein).

The surfactant(s) are suitably selected from Polysorbate 20(Polyoxyethylene (20) sorbitan monolaurate), Polysorbate 40(Polyoxyethylene (20) sorbitan monopalmitate), Polysorbate 60(Polyoxyethylene (20) sorbitan monostearate), Polysorbate 80(Polyoxyethylene (20) sorbitan monooleate), Sorbitan monolaurate,Sorbitan monopalmitate, Sorbitan monostearate, Sorbitan tristearate,and/or Sorbitan monooleate.

In a particular embodiment, the surfactant(s) are selected fromPolysorbate 20, Polysorbate 40, Polysorbate 60, and/or Polysorbate 80.In a particular embodiment, the liquid pharmaceutical compositioncomprises a single surfactant selected from Polysorbate 20, Polysorbate40, Polysorbate 60, and Polysorbate 80.

In a particular embodiment, the surfactant is polysorbate 80 orpolysorbate 20. In a particular embodiment, the surfactant ispolysorbate 80.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) at a concentration of fromabout 0.0001 to about 5 mM (i.e. 0.1 μM-5 mM), more suitably from about0.001 to about 2 mM, more suitably from about 0.01 to about 1.0 mM. Inan embodiment, the surfactant(s) is present at a concentration ofbetween 0.72 and 0.80 mM, most suitably about 0.76 mM. In an embodiment,polysorbate 80 is present at a concentration of 0.76 mM.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) at a concentration of fromabout 0.001 mg/mL to about 5 mg/mL, more suitably from about 0.01 mg/mLto about 2 mg/mL, more suitably from about 0.05 mg/mL to about 1.5mg/mL. In an embodiment, the surfactant(s) is present at a concentrationof between 0.9 mg/mL and 1.1 mg/mL, most suitably about 1.0 mg/mL. In aparticular embodiment, polysorbate 80 is present at a concentration ofabout 1.0 mg/mL.

Suitably, the liquid pharmaceutical composition comprises thesurfactant(s) (most suitably polysorbate 80) in a molar ratio ofsurfactant(s) to adalimumab of from about 1:3500 to about 15:1, moresuitably from about 1:350 to about 6:1, more suitably from about 1:35 toabout 3:1. In an embodiment, the surfactant(s) is present at a molarratio of surfactant(s) to adalimumab of from about 2.1:1 to about 2.3:1,most suitably about 2.2:1. In an embodiment, polysorbate 80 is presentat a molar ratio of polysorbate 80 to adalimumab of about 2.2:1.

As illustrated in the Example section, liquid pharmaceuticalcompositions of the invention including a surfactant as defined hereinperform particularly well in stress tests, especially in relation toaggregation, fragmentation and protein unfolding, which can be importantindicators of stability and drug product viability. Furthermore, liquidpharmaceutical compositions comprising polysorbate, particularly in anamount range as stipulated, perform particularly well.

Other Parameters Relating to the Invention

Osmolality

Suitably, the osmolality of the liquid pharmaceutical composition isbetween 200 and 400 mOsm/kg, more suitably between 220 and 390 mOsm/kg,more suitably between 230 and 350 mOsm/kg, more suitably between 240 and340 mOsm/kg, more suitably between 260 and 320 mOsm/kg, most suitablybetween 280 and 310 mOsm/kg. Suitably the relative amounts andconcentrations of the various components of the composition may bejudiciously tuned to achieve the desired osmolality, and the particularnovel combination of components allows this to be largely achievedwithout undermining other important parameters. However, suitably therelative amounts and concentrations of the various components of thecomposition may be selected so as to optimise other parameters—thepresent disclosure, including the examples and protocols set forththerein, enable the skilled person to achieve this end and to realise a,some, or all of the benefits of the present invention.

Protein Unfolding Temperature

Suitably, the protein unfolding temperature (suitably as measured viathe DSF protocols defined herein) of adalimumab in the liquidpharmaceutical composition of the invention is greater than or equal to65° C., more suitably greater than or equal to 70° C. The novelcombination of components present within the composition of theinvention enables the skilled person to achieve high unfoldingtemperatures, which may be considered desirable from a thermal stabilityperspective.

Parameters when Subjected to Thermal Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than factor of3, suitably by no more than factor of 2.5, suitably by no more thanfactor of 2.2.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than factor of3, suitably by no more than factor of 2.5, suitably by no more thanfactor of 2.2.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is thermally stressed at 40° C. (i.e. the composition ismaintained at a temperature of 40° C.) over a period of 28 days,suitably by no more than a factor of 1.5, suitably by no more than afactor of 1.2, and suitably the turbidity does not increase at all.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is thermallystressed at 40° C. (i.e. the composition is maintained at a temperatureof 40° C.) over a period of 28 days, suitably by no more than 0.2 pHunits, suitably by no more than 0.1 pH units, most suitably the pH doesnot change at all (to one decimal place).

Parameters when Subjected to Mechanical Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 2 (i.e. 2 times the amountrelative to an arbitrary start time) when the composition ismechanically stressed (i.e. shaken as per the protocols outlined herein)over a period of 48 hours, suitably by no more than factor of 1.5,suitably by no more than factor of 1.2, suitably by no more than factorof 1.1.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 2 (i.e. 2 times the amountrelative to an arbitrary start time) when the composition ismechanically stressed (i.e. shaken as per the protocols outlined herein)over a period of 48 hours, suitably by no more than factor of 1.5,suitably by no more than factor of 1.2, suitably by no more than factorof 1.1.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is mechanically stressed (i.e. shaken as per the protocolsoutlined herein) over a period of 48 hours, suitably by no more thanfactor of 1.5, suitably by no more than factor of 1.2, suitably by nomore than factor of 1.1, and suitably the turbidity does not increase atall.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is mechanicallystressed (i.e. shaken as per the protocols outlined herein) over aperiod of 48 hours, suitably by no more 0.2 pH units, suitably by nomore than 0.1 pH units, most suitably the pH does not change at all (toone decimal place).

Parameters when Subjected to Light Stress

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 50 (i.e. 50 times the amountrelative to an arbitrary start time) when the composition is lightstressed (i.e. the composition is exposed to light in accordance withprotocols disclosed herein, i.e. 7 hours at 765 W/m²), suitably by nomore than factor of 45, suitably by no more than factor of 35, suitablyby no more than factor of 30.

Suitably the quantity (or concentration) of fragments (suitably derivedfrom adalimumab and suitably measured via the bioanalyzer protocolsdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 4 (i.e. 4 times the amountrelative to an arbitrary start time) when the composition is lightstressed (i.e. the composition is exposed to light in accordance withprotocols disclosed herein, i.e. 7 hours at 765 W/m²), suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.

Suitably the turbidity (suitably as measured via nephelometry inaccordance with the protocols set forth herein) of the liquidpharmaceutical composition increases by no more than a factor of 2 (i.e.2 times the amount relative to an arbitrary start time) when thecomposition is light stressed (i.e. the composition is exposed to lightin accordance with protocols disclosed herein, i.e. 7 hours at 765W/m²), suitably by no more than a factor of 1.5, suitably by no morethan a factor of 1.2, and suitably the turbidity does not increase atall.

Suitably the pH of the liquid pharmaceutical composition changes(whether through increase or decrease, though generally by a decrease inpH) by no more than 0.5 pH units when the composition is light stressed(i.e. the composition is exposed to light in accordance with protocolsdisclosed herein, i.e. 7 hours at 765 W/m²), suitably by no more than0.2 pH units, suitably by no more than 0.1 pH units, most suitably thepH does not change at all (to one decimal place).

Suitably the isoform profile of adalimumab, particularly the integratedarea of the “main peak”, in the liquid pharmaceutical composition(suitably measured via isoelectronic focussing, suitably cIEF, suitablyusing an iCE280, suitably employing a protocol as set forth herein) isreasonably stable when the composition is light stressed (i.e. thecomposition is exposed to light in accordance with protocols disclosedherein, i.e. 7 hours at 765 W/m²). Suitably, light stressing isperformed in accordance with current ICH Q1B guidelines of the EuropeanMedicines Agency (in relation to photostability testing of new activesubstances and medicinal products), suitably as illustrated by documentCPMP/ICH/279/95. Suitably the isoform profile of the adalimumab withinthe composition, as measured by reference to the integrated area of the“main peak” relating to the adalimumab in an electropherogram producedby isoelectronic focussing (suitably capillary isoelectronic focussingas described herein, or optionally other standard isoelectronicfocussing protocols well known in the art), changes by no more than 20%when subjected to light stress (suitably 7 hours exposure to 765 W/m²light, suitably in accordance with current ICH Q1B guidelines of theEuropean Medicines Agency, suitably document CPMP/ICH/279/95),especially where an adalimumab biosimilar is used. Suitably, the isoformprofile of adalimumab within the composition (suitably measured byreference to the integrated area of the “main peak” relating toadalimumab) does not change by more than 15% (whether an increase orreduction in peak area) when light stressed in this manner (especiallywhere an adalimumab biosimilar is used), suitably by no more than 10%,suitably by no more than 5%, suitably by no more than 4%, suitably by nomore than 3%. Prior art adalimumab compositions exhibit inferior isoformprofile stability due to photo-oxidation phenomenon which is dulyinhibited through using an acetate buffer within the particular liquidpharmaceutical compositions of the invention. As such, in the liquidpharmaceutical compositions of the invention adalimumab exhibitssuperior photostability. In a particular embodiment, adalimumab has aphotostability in the liquid pharmaceutical composition of the inventiongreater than commercial HUMIRA® formulations as defined herein (suitablyas indicated by the relative isoform profiles, particularly in relationto the adalimumab main peak), suitably even where adalimumab biosimilarsare used. It will be understood that the “main peak” corresponding toadalimumab refers to the main adalimumab peak of an electropherogram(i.e. that with the highest integrated peak area) resulting fromisoelectronic focussing measurements, suitably performed as definedherein. Suitably the electropherogram is acquired at 280 nm, suitablyover pre-focusing and focusing times of 1 and 6 minutes respectively,suitably at a voltage of 1500 V (pre-focusing) and 3000 V (focusing).Suitably the peaks are absorbance peaks, suitably at 280 nm. Theseparation of the various isoforms is suitably achieved using 100 mMsodium hydroxide (in 0.1% methyl cellulose) as a cathodic solution andsuitably 80 mM o-phosphoric acid (in 0.1% methyl cellulose) as an anodicsolution. Suitably samples for isoelectronic focussing measurements areprepared according to a protocol defined herein or elsewhere in the art,but in particular may suitably involve one or more or all of: i)purification; ii) removal of salts (e.g. with centrifugation, suitablywith a cut off at 10 kDa); iii) pre dilution to give a protein contentof approximately 5.0 mg/mL, suitably approximately 1.0 mg/mL, whereinthe diluent may optionally include methyl cellulose, Pharmalyte 5-8 (GEHealthcare), Pharmalyte 8-10.5 (GE Healthcare), low pI marker 7.05(Protein Simple), high pI marker 9.50 (Protein Simple) and purifiedwater; iv) one or more additional centrifugation steps (e.g. 3 mins at10000 rpm, suitably followed by 2 mins at 7000 rpm, suitably on a 150micolitre sample). The isoelectronic focussing is suitably capillaryisoelectron focussing (cIEF), and is suitably performed using an iCE280system by Protein Simple.

Parameters when Subjected to Freeze/Thaw Cycles

Suitably the quantity (or concentration) of aggregates (suitably derivedfrom adalimumab, and suitably as determined by the SE-HPLC protocols asdefined herein) present within the liquid pharmaceutical compositionincreases by no more than a factor of 1.5 (i.e. 1.5 times the amountrelative to an arbitrary start time) when the composition is subjectedto five freeze/thaw cycles (i.e. the composition is frozen and thawedfive times in accordance with protocols disclosed herein, i.e. −80° C.to 20° C. five times), suitably by no more than factor of 1.2, suitablyby no more than factor of 1.1, suitably by there is (substantially) noincrease at all in the quantity (or concentration) of aggregates.

Suitably the quantity (or concentration) of sub-visible particles orprecipitates, with a particle size less than or equal to 25 microns,present within the liquid pharmaceutical composition increases by nomore than a factor of 4 (i.e. 4 times the amount relative to anarbitrary start time) when the composition is subjected to fivefreeze/thaw cycles (i.e. the composition is frozen and thawed five timesin accordance with protocols disclosed herein, i.e. −80° C. to 20° C.five times), suitably by no more than factor of 3, suitably by no morethan factor of 2.5, suitably by no more than factor of 2.2. Suitably thequantity (or concentration) of sub-visible particles or precipitates,with a particle size less than or equal to 10 microns, present withinthe liquid pharmaceutical composition increases by no more than a factorof 4 (i.e. 4 times the amount relative to an arbitrary start time) whenthe composition is subjected to five freeze/thaw cycles (i.e. thecomposition is frozen and thawed five times in accordance with protocolsdisclosed herein, i.e. −80° C. to 20° C. five times), suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.2.

Suitably the quantity (or concentration) of sub-visible particles orprecipitates, with a particle size less than or equal to 25 microns,present within the liquid pharmaceutical composition increases by nomore than a factor of 4 (i.e. 4 times the amount relative to anarbitrary start time) when the composition is subjected to 5 freeze/thawcycles, suitably by no more than factor of 3, suitably by no more thanfactor of 2.5, suitably by no more than factor of 2.2. Suitably thequantity (or concentration) of sub-visible particles or precipitates,with a particle size less than or equal to 10 microns, present withinthe liquid pharmaceutical composition increases by no more than a factorof 4 (i.e. 4 times the amount relative to an arbitrary start time) whenthe composition is subjected to 5 freeze/thaw cycles, suitably by nomore than factor of 3, suitably by no more than factor of 2.5, suitablyby no more than factor of 2.2.

Methods of Stabilising Antibody

In view of the aforementioned points in this sub-section, and the datapresented in the examples, the present invention also provides a methodof stabilising liquid adalimumab compositions (chemically and/orphysically optionally in relation to any one or more of theaforementioned parameters/properties), comprising mixing with adalimumabwith any relevant components required to form a liquid pharmaceuticalcomposition as defined herein. Different embodiments will suitablyrequire different combinations of components to be mixed, potentially indifferent amounts, and the skilled person can readily deduce suchcombinations and amounts by reference to the foregoing disclosurerelating to the liquid pharmaceutical composition. Such differentcombinations of components may stabilise liquid adalimumab compositionsin different respects. For instance, mixing adalimumab with theaforementioned components to form a liquid pharmaceutical composition asdefined herein may stabilise adalimumab by:

-   i) Increasing the protein unfolding temperature of adalimumab;-   ii) Inhibiting the formation of aggregates;-   iii) Inhibiting the formation of fragments;-   iv) Inhibiting the formation of sub-visible particles (either ≤25    microns or ≤10 microns);-   v) Inhibiting turbidification;-   vi) Inhibiting pH changes;-   vii) Inhibiting photo-oxidation; and/or-   viii) Reducing instability upon freeze/thaw cycles.

As such, the present invention provides a method of achieving one, some,or all of the following benefits:

-   i) Increased protein unfolding temperatures for adalimumab;-   ii) Inhibition of formation of aggregates;-   iii) Inhibition of formation of fragments;-   iv) Inhibition of formation of sub-visible particles (either ≤25    microns or ≤10 microns);-   v) Inhibition of turbidification;-   vi) Inhibition of pH changes;-   vii) Inhibition of photo-oxidation;-   viii) Reduced instability upon freeze/thaw cycles; and/or-   ix) Stabilisation of the isoform profile (especially with respect to    the “main peak” as defined herein);    the method comprising manufacturing a liquid pharmaceutical    composition of adalimumab as defined herein.

Suitably, the liquid pharmaceutical compositions of the invention have ashelf life of at least 6 months, suitably at least 12 months, suitablyat least 18 months, more suitably at least 24 months. Suitably, theliquid pharmaceutical compositions of the invention have a shelf life ofat least 6 months, suitably at least 12 months, suitably at least 18months, more suitably at least 24 months, at a temperature of 2-8° C.

Enabling the Skilled Person to Optimise Key Stability Properties

The novel combination of components disclosed for use in liquidpharmaceutical compositions of the invention enables the skilled personto produce (and judiciously fine-tube) compositions which exhibitcomparable or enhanced properties relative to compositions of the priorart. In particular, the present disclosure now provides the skilledperson with all the necessary tools to optimise formulation stability,and in particular optimise one or more of: inhibition of aggregation,fragmentation, protein unfolding, precipitation, pH slippage, andoxidation (especially photo-oxidation). Furthermore, the skilled personis given guidance on how to achieve such optimisations (throughjudiciously varying the compositions) and how, in the process, tominimise any detrimental side-effects. The present disclosure enablesthe skilled person to work across the scope of the invention to producea variety of specific compositions which exhibit comparable or improvedproperties relative to compositions of the prior art, and this can beachieved using fewer components.

Particular Embodiments

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an acetate buffering agent (e.g. sodium acetate) (or an acetate        buffer system);    -   a sugar stabiliser (e.g. trehalose); and    -   a surfactant (e.g. polysorbate 80).

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an acetate buffering agent (e.g. sodium acetate) (or an acetate        buffer system);    -   a sugar stabiliser (e.g. trehalose);    -   a tonicifier (e.g. sodium chloride); and    -   optionally a surfactant (e.g. polysorbate 80).

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffer system, and a sugar stabiliser in a molarratio of 1:14-40:288-865 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate buffer system,sugar stabiliser, and a tonicifier in a molar ratio of1:14-40:288-865:28-576 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate buffer system,sugar stabiliser, tonicifier, and surfactant in a molar ratio of1:14-40:288-865:28-576:0.1-3.2 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffer system, and a sugar stabiliser in a molarratio of 1:14-40:548-605 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate buffer system,sugar stabiliser, and a tonicifier in a molar ratio of1:14-40:548-605:115-173 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate buffer system,sugar stabiliser, tonicifier, and surfactant in a molar ratio of1:14-40:548-605:115-173:2-2.4 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, sodium acetate/acetic acid buffer system, and trehalose in amolar ratio of 1:5.7-145:288-865 respectively. In an embodiment, theliquid pharmaceutical composition comprises adalimumab, sodiumacetate/acetic acid buffer system, trehalose, and sodium chloride in amolar ratio of 1:5.7-145:288-865:28-576 respectively. In an embodiment,the liquid pharmaceutical composition comprises adalimumab, sodiumacetate/acetic acid buffer system, trehalose, sodium chloride, andpolysorbate 80 in a molar ratio of 1:5.7-145:288-865:28-576:0.002-11respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, sodium acetate/acetic acid buffer system, and trehalose in amolar ratio of 1:14-40:548-605 respectively. In an embodiment, theliquid pharmaceutical composition comprises adalimumab, sodiumacetate/acetic acid buffer system, trehalose, and sodium chloride in amolar ratio of 1:14-40:548-605:115-173 respectively. In an embodiment,the liquid pharmaceutical composition comprises adalimumab, sodiumacetate/acetic acid buffer system, trehalose, sodium chloride, andpolysorbate 80 in a molar ratio of 1:14-40:548-605:115-173:2-2.4respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, sodium acetate/acetic acid buffer system, and trehalose in amolar ratio of 1:28.8:576 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, sodium acetate/aceticacid buffer system, trehalose, and sodium chloride in a molar ratio of1:28.8:576:144 respectively. In an embodiment, the liquid pharmaceuticalcomposition comprises adalimumab, sodium acetate/acetic acid buffersystem, trehalose, sodium chloride, and polysorbate 80 in a molar ratioof 1:28.8:576:144:2.2 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffering species, and trehalose in a weight ratioof 25-75:0.12-3.0:15-140 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate bufferingspecies, trehalose, and sodium chloride in a weight ratio of25-75:0.12-3.0:15-140:0.5-12 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate bufferingspecies, trehalose, sodium chloride, and polysorbate 80 in a weightratio of 25-75:0.12-3.0:15-140:0.5-12:0.01-2 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffering species, and trehalose in a weight ratioof 45-55:0.30-0.84:65-72 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate bufferingspecies, trehalose, and sodium chloride in a weight ratio of45-55:0.30-0.84:65-72:2.7-3.1 respectively. In an embodiment, the liquidpharmaceutical composition comprises adalimumab, acetate bufferingspecies, trehalose, sodium chloride, and polysorbate 80 in a weightratio of 45-55:0.30-0.84:65-72:2.7-3.1:0.9-1.1 respectively.

In an embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffering species, and trehalose in a weight ratioof 50:0.6:68 respectively. In an embodiment, the liquid pharmaceuticalcomposition comprises adalimumab, acetate buffering species, trehalose,and sodium chloride in a weight ratio of 50:0.6:68:2.9 respectively. Inan embodiment, the liquid pharmaceutical composition comprisesadalimumab, acetate buffering species, trehalose, sodium chloride, andpolysorbate 80 in a weight ratio of 50:0.6:68:2.9:1 respectively.

Any of the aforementioned embodiments relating to molar and/or weightratios of the various components may be additionally defined byreference to the (substantial or entire) absence or low levels ofcomponent(s) such as arginine, amino acids, surfactants (optionally withthe exception of polysorbate 80), and/or phosphate bufferingagents/systems, as defined anywhere herein.

It will be appreciated that the buffering agent (e.g. sodium acetate) orbuffer system (e.g. sodium acetate/acetic acid) of any of theaforementioned embodiments may be directly incorporated into thecompositions or may be produced in situ, for instance, via an acid basereaction, suitably by reacting a conjugate acid of the buffering agent(e.g. acetic acid) with a base (e.g. sodium hydroxide). Regardless ofthe method used to provide or produce the buffering agent or buffersystem, suitably the resulting composition ultimately comprises anappropriate balance of the buffering agent and any acid/base conjugateto furnish the desired pH. The skilled person will be readily able tocalculate or experimentally determine, without undue effort, theappropriate balance of buffering agent and acid/base conjugate, and/orthe amount of base which needs to be added to a conjugate acid in orderto produce the appropriate amount of buffering agent and furnish thedesired pH.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   adalimumab;    -   an acetate buffering agent (e.g. sodium acetate) (or acetate        buffer system);    -   a sugar stabiliser (e.g. trehalose);    -   a tonicifier (e.g. sodium chloride);    -   optionally a surfactant (e.g. polysorbate 80); and    -   water (for injection);    -   wherein the composition:        -   is (substantially or entirely) free of arginine (suitably            L-arginine); comprises arginine in a concentration of at            most 0.1 mM;        -   is (substantially or entirely) free of amino acids or            comprises one or more amino acids in a (collective)            concentration of at most 0.1 mM;        -   is (substantially or entirely) free of surfactants with the            optional exception of polysorbate 80 or comprises one or            more of said surfactants (optionally excluding            polysorbate 80) in a (collective) concentration of at most 1            mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   Adalimumab (suitably in a concentration as defined herein);    -   5 to 14 mM acetate buffer system (e.g. sodium acetate/acetic        acid);    -   100 to about 300 mM a sugar stabiliser (e.g. trehalose);    -   10 to about 200 mM a tonicifier (e.g. sodium chloride);    -   (optionally) 0.05 mg/mL to about 1.5 mg/mL surfactant (e.g.        polysorbate 80); and    -   water (for injection);    -   wherein the composition:        -   has a pH between 5.0 and 6.7 (e.g. pH 5.2)        -   is (substantially or entirely) free of arginine (suitably            L-arginine); comprises arginine in a concentration of at            most 0.1 mM;        -   is (substantially or entirely) free of amino acids or            comprises one or more amino acids in a (collective)            concentration of at most 0.1 mM;        -   is (substantially or entirely) free of surfactants with the            optional exception of polysorbate 80 or comprises one or            more of said surfactants (optionally excluding            polysorbate 80) in a (collective) concentration of at most 1            mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   25 to about 75 mg/mL adalimumab;    -   2 to about 50 mM sodium acetate/acetic acid buffer system;    -   100 to about 300 mM trehalose;    -   10 to about 200 mM sodium chloride;    -   0.001 mg/mL to about 5 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition:        -   has a pH between 5.0 and 5.5;        -   is (substantially or entirely) free of arginine (suitably            L-arginine) or comprises arginine in a concentration of at            most 0.1 mM;        -   is (substantially or entirely) free of amino acids or            comprises one or more amino acids in a (collective)            concentration of at most 0.1 mM;        -   is (substantially or entirely) free of surfactants with the            exception of polysorbate 80 or comprises one or more of said            surfactants (excluding polysorbate 80) in a (collective)            concentration of at most 1 mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.1 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   45 to about 55 mg/ml adalimumab;    -   5 to 14 mM sodium acetate/acetic acid buffer system;    -   190 to 210 mM trehalose;    -   40 to 60 mM sodium chloride;    -   0.9 mg/mL and 1.1 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition:        -   has a pH between 5.1 and 5.3;        -   is (substantially or entirely) free of arginine (suitably            L-arginine) or comprises arginine in a concentration of at            most 0.001 mM;        -   is (substantially or entirely) free of amino acids or            comprises one or more amino acids in a (collective)            concentration of at most 0.001 mM.        -   is (substantially or entirely) free of surfactants with the            exception of polysorbate 80 or comprises one or more of said            surfactants (excluding polysorbate 80) in a (collective)            concentration of at most 0.0001 mM; and/or        -   is (substantially or entirely) free of phosphate buffering            agents (e.g. sodium dihydrogen phosphate, disodium hydrogen            phosphate) or comprises a phosphate buffer system in a            concentration of at most 0.001 mM.

In an embodiment, the liquid pharmaceutical composition comprises:

-   -   50 mg/ml adalimumab;    -   10 mM sodium acetate/acetic acid buffer system;    -   200 mM trehalose;    -   50 mM sodium chloride;    -   1.0 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition:        -   has a pH of 5.2;        -   is free of arginine;        -   is free of amino acids;        -   is free of surfactants with the exception of polysorbate 80;            and        -   is free of phosphate buffering agents/systems.

Preferably, the liquid pharmaceutical composition consists essentiallyof:

-   -   25 to about 75 mg/mL adalimumab;    -   2 to about 50 mM sodium acetate/acetic acid buffer system;    -   100 to about 300 mM trehalose;    -   10 to about 200 mM sodium chloride;    -   0.001 mg/mL to about 5 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition has a pH between 5.0 and 5.5.

More preferably, the liquid pharmaceutical composition consistsessentially of:

-   -   40 to about 60 mg/mL adalimumab;    -   5 to about 15 mM sodium acetate/acetic acid buffer system;    -   175 to about 225 mM trehalose;    -   25 to about 75 mM sodium chloride;    -   0.5 mg/mL to about 1.5 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition has a pH between 5.1 and 5.3.

Ideally, the liquid pharmaceutical composition consists essentially of:

-   -   50 mg/mL adalimumab;    -   10 mM sodium acetate/acetic acid buffer system;    -   200 mM trehalose;    -   50 mM sodium chloride;    -   1 mg/mL polysorbate 80; and    -   water (for injection);        -   wherein the composition has a pH of 5.2.

Suitably, the liquid pharmaceutical composition may be as set forth inany of the preceding embodiments, except that the absence or low levelsof component(s) such as arginine, amino acids, surfactants (optionallywith the exception of polysorbate 80), and phosphate bufferingagents/systems, rather than being defined by reference toconcentration(s) (i.e. molarity) may instead be defined by reference tocorresponding molar ratios of the component to buffer system;corresponding weight ratios of the component to adalimumab; orcorresponding molar ratios of the component to adalimumab. The skilledperson will readily deduce for each component, from the relevant sectionof this specification relating to that specific component, which molarand weight ratios correspond to which concentrations, since herein therelevant molar and weight ratios are listed to respectively correspondto given concentrations. For example, in the case of arginine, theoptional concentrations of “at most 0.1 mM, more suitably at most 0.01mM, most suitably at most 0.001 mM” respectively correspond with a molarratio of arginine to buffer system of “at most 1:150 . . . more suitablyat most 1:1500, most suitably at most 1:15,000”; with “a weight ratio ofarginine to adalimumab of at most 1:3000 . . . more suitably at most1:30,000, most suitably at most 1:300,000”; and with a molar ratio ofarginine to adalimumab of at most 1:3.75 . . . more suitably at most1:37.5, most suitably at most 1:375”. The same correspondences apply foramino acids, surfactants, and phosphate buffering agents/systems.

Method of Manufacturing a Liquid Pharmaceutical Composition

The present invention provides a method of manufacturing a liquidpharmaceutical composition, suitably as defined herein. The methodsuitably comprises mixing together, in any particular order deemedappropriate, any relevant components required to form a liquidpharmaceutical composition as defined herein. The skilled person mayrefer to the Examples or techniques well known in the art for formingliquid pharmaceutical compositions (especially those for injection viasyringe). Different embodiments will suitably require differentcombinations of components to be mixed, potentially in differentamounts. The skilled person can readily deduce such combinations andamounts by reference to the foregoing disclosure relating to the liquidpharmaceutical composition.

Suitably the method involves mixing together the relevant componentssuitably, in a diluent (e.g. water), suitably so that all of thecomponents are (substantially or entirely) dissolved in the diluent.

The method may involve first preparing a pre-mixture (or pre-solution)of some or all components (optionally with some or all of the diluent)excluding adalimumab, and adalimumab may then itself (optionally with orpre-dissolved in some of the diluent) be mixed with the pre-mixture (orpre-solution) to afford the liquid pharmaceutical composition, or acomposition to which final components are then added to furnish thefinal liquid pharmaceutical composition. Most suitably, the pre-mixturecontains all components except for the adalimumab and optionally alsosome diluent (which may be used to pre-dissolve adalimumab), suitably sothat adalimumab is added to a mixture which offers optimal stabilisationof adalimumab. Suitably the aforementioned pre-mixture is prepared withthe desired pH for the final liquid pharmaceutical formulation.

Suitably, the method involves forming a buffer system, suitably a buffersystem comprising a buffering agent as defined herein. The buffer systemis suitably formed in a pre-mixture prior to the addition of adalimumab,though the buffer system may optionally be formed with adalimumabpresent. The buffer system may be formed through simply mixing thebuffering agent (supplied ready-made) with its acid/base conjugate(suitably in appropriate relative quantities to provide the desiredpH—this can be determined by the skilled person either theoretically orexperimentally). In the case of an acetate buffer system, this meansmixing sodium acetate with acetic acid. Alternatively, the buffer systemmay be formed through adding a strong acid (e.g. HCl) to the bufferingagent (e.g. sodium acetate) in order to form in situ the acid/baseconjugate (e.g. acetic acid) (again suitably in appropriate relativequantities to provide the desired pH). Alternatively, the buffer systemmay be formed through adding a strong base (e.g. sodium hydroxide) tothe acid/base conjugate (e.g. acetic acid) of the buffering agent (e.g.sodium acetate) in order to form in situ the buffering agent (againsuitably in appropriate relative quantities to provide the desired pH).The pH of either the pre-mixture of final liquid pharmaceuticalcomposition may be judiciously adjusted by adding the required quantityof strong base or strong acid, or even a quantity of buffering agent oracid/base conjugate.

In certain embodiments, the buffering agent and/or buffer system ispre-formed as a separate mixture, and the buffer system is transferredto a precursor of the liquid pharmaceutical composition (comprising someor all components save for the buffering agent and/or buffer system,suitably comprising adalimumab and potentially only adalimumab) viabuffer exchange (e.g. using diafiltration until the relevantconcentrations or osmolality is reached). Additional excipients may beadded thereafter if necessary in order to produce the final liquidpharmaceutical composition. The pH may be adjusted once or before allthe components are present.

Any, some, or all components may be pre-dissolved or pre-mixed with adiluent prior to mixing with other components.

The final liquid pharmaceutical composition may be filtered, suitably toremove particulate matter. Suitably filtration is through filters sizedat or below 1 μm, suitably at 0.22 μm. Suitably, filtration is througheither PES filters or PVDF filters, suitably with 0.22 μm PES filters.

The present invention also provides a liquid pharmaceutical compositionobtainable by, obtained by, or directly obtained by the method ofmanufacture herein described.

Drug-Delivery Device

The present invention provides a drug delivery device comprising aliquid pharmaceutical composition as defined herein. Suitably the drugdelivery device comprises a chamber within which the pharmaceuticalcomposition resides. Suitably the drug delivery device is sterile.

The drug delivery device may a vial, ampoule, syringe, injection pen(e.g. essentially incorporating a syringe), or intravenous bag. Mostsuitably the drug delivery device is a syringe, suitably an injectionpen. Suitably the syringe is a glass syringe. Suitably the syringecomprises a needle, suitably a 29 G ½″ needle.

The present invention provides a method of manufacturing a drug deliverydevice, suitably as defined herein, the method comprising incorporatinga liquid pharmaceutical composition as defined herein within a drugdelivery device. Such manufacture typically involves charging the liquidpharmaceutical composition as defined herein to a syringe, suitably viaa needle affixed thereto. The needle may thereafter be removed,replaced, or remain.

According to an eleventh aspect of the present invention there isprovided a drug delivery device obtainable by, obtained by, or directlyobtained by a method of manufacture defined herein.

Package

The present invention provides a package comprising a liquidpharmaceutical composition as defined herein. Suitably the packagecomprises a drug delivery device as defined herein, suitably a pluralityof drug delivery devices. The package may comprise any suitablecontainer for containing one or more drug delivery devices.

The present invention provides a method of manufacturing a package, themethod comprising incorporating a liquid pharmaceutical composition asdefined herein within a package. Suitably this is achieved byincorporating said liquid pharmaceutical composition within one or moredrug delivery devices, and thereafter incorporating the one or morepre-filled drug delivery devices into a container present within thepackage.

The present invention provides a package obtainable by, obtained by, ordirectly obtained by a method of manufacture defined herein.

Kit of Parts

The present invention provides a kit of parts comprising a drug deliverydevice (without the liquid pharmaceutical composition incorporatedtherein), a liquid pharmaceutical composition as defined herein(optionally contained in a separate package or container), andoptionally a set of instructions with directions regarding theadministration (e.g. sub-cutaneous) of the liquid pharmaceuticalcomposition. The user may then fill the drug delivery device with theliquid pharmaceutical composition (which may be provided in a vial orampoule or such like) prior to administration.

Uses of Pharmaceutical Liquid Composition and Methods of Treatment

According to a twelfth aspect of the present invention there is provideda method of treating a disease or medical disorder; a liquidpharmaceutical composition for use in therapy; a use of a liquidpharmaceutical composition in the manufacture of a medicament for thetreatment of a disease or disorder; a method of treating a tumournecrosis factor-alpha (TNF-α)-related autoimmune disease; a liquidpharmaceutical composition for use in the treatment of a tumour necrosisfactor-alpha (TNF-α)-related autoimmune disease; a use of a liquidpharmaceutical composition in the manufacture of a medicament for thetreatment of a tumour necrosis factor-alpha (TNF-α)-related autoimmunedisease; a method of treating rheumatoid arthritis, psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis and/or juvenile idiopathic arthritis; a liquidpharmaceutical composition for use in the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis; and a use of a liquid pharmaceutical compositionin the manufacture of a medicament for the treatment of rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease,ulcerative colitis, moderate to severe chronic psoriasis and/or juvenileidiopathic arthritis; as defined herein.

The liquid pharmaceutical compositions defined herein may be used totreat any one or more of the aforementioned diseases or medicaldisorders. In a particular embodiment, the liquid pharmaceuticalcompositions are used to treat rheumatoid arthritis, Crohn's disease andpsoriasis.

The liquid pharmaceutical compositions are suitably parenterallyadministered, suitably via sub-cutaneous injection.

EXAMPLES

Materials and Equipment

The following materials were used in the preparation of formulationsdescribed in the Examples that follow:

Ingredient Acetic acid (glacial) 100% Adalimumab DS Argininemonohydrochloride Aspartic Acid Citric Acid Monohydrate Dibasicsodiumphosphate dihydrate Lysine hydrochloride Mannitol Monobasicsodiumphosphate dihydrate Poloxamer 188 Polysorbate 80 Sodium chloride Sodiumcitrate Sodium hydroxide solution 30% Trehalose dihydrate WFI

The following disposable equipment and materials were used in theExamples and Screen Experiments which follow.

Item Code Supplier EppendorfTubes NA Eppendorf (0.5 mL, 1.5 mL, 2.0 mL)Falcon 352096 (15 mL), 352070 (50 mL) Becton polypropylene tubesDickinson PES membrane MillexGP Express PES membrane REF Millipore (0.22μm) SLGP033RS filter unit PETG bottles 3420-1000, 3420-0500, 2019-0250,Nalgene 3420-0125, 3420-0060, 2019-0030

The following packaging was used in the Examples and Screen Experimentswhich follow.

Item Code Supplier DIN2R Type I glass 0212060.6112 11200000A Nuova Ompivial 1 mL stopper S2-F451 RSV; D 21-7S RB2-40 Daikyo Seiko, LTD 13 mmflip-off cap 12000350 MS-A

The following equipment was used in the Examples and Screen Experimentswhich follow.

Item Mod. Manufacturer Analytical scales AX205, PG2002-S Mettler ToledoBenchtop xenon Suntest CPS+ Atlas instrument Calibrated pipettes P20,P100, P200, P1000 Gilson HPLC Alliance Waters iCE280 Fast IEF AnalyzerConvergent Bioscience Osmometer Osmomat 030/D Gonotec PCR 7500 FastReal-Time AB Applied Biosystem pH meters Seven Multi Mettler ToledoRefrigerators +2-8° C. Angelantoni Software Design Expert ver. 7.1.5Stat-Ease, Inc. Thermostatic cabinets +25° C., +40° C. AngelantoniTurbidimeter 2100AN IS Hach Lange UV Spectrophotometer Lambda 35 PerkinElmerAnalytical Techniques and Protocols

The following analytical methods of protocols were employed, in theExamples and Screening Experiments which follow, for the reasons statedin the table below:

Method No. Analytical Method Scope of the test 1 Bioanalyzer Purity 2DSF Unfolding temperature 3 iCE280 Isoforms profile 4 OD Protein Content5 SE-HPLC Aggregates determination 6 Nephelometry Turbidity 7 OsmolalityOsmolality of solution 8 pH pH determination 9 Sub-visible particlesParticle count

The individual protocols for each of the above analytical methods aredescribed in turn below, and references in the Examples and ScreeningExperiments to any such analytical methods used these protocols.

1. Purity—Bioanalyzer

A 2100 Bioanalyzer was used. Protocols can be found in the relevantinstruction manual. However, the protocols have been additionallyrefined as follows.

Solutions:

Gel-Dye Mix (Staining Solution):

Add 25 μL of 230plus dye concentrate to a protein 230plus gel matrixtube. Vortex well, and spin down the tube for 15 seconds. Transfer to aspin filter and centrifuge at 2500 rpm for at least 20 min. The solutionis ready to use. Store the solution at +5±3° C. for not more than 4weeks.

Destaining Solution:

Pipette 650 μL of gel matrix into a spin filter. Centrifuge at 2500 rpmfor at least 25 min. Store the solution at +5±3° C. for not more than 4weeks.

Sample Buffer:

It is recommended to divide the 200 μL of sample buffer into aliquots of25 μL and defreeze aliquot for each chip. Store the Sample buffer stocksolution and the aliquots at −20° C., not longer than the expiring dateprovided by the supplier.

Maleimide Stock Solution:

Dissolve 23.4 mg of Male imide in 1 mL MilliQ water (O.24M). Vortex wellthe solution. Subsequently dilute the solution 1:4 with MilliQ water.(e.g. 50 μL Stock Sol.+150 μL MilliQ). The final concentration of thediluted Maleimide solution is 60 mM. (Since no data is available yet onthe stability of this solution, it must be prepared freshly beforestarting each analytical session).

OTf-Solution:

For the analysis of Adalimumab samples the reducing solution must beprepared with 1M DTT, therefore dissolve 154.0 mg DTT in 1 mL MilliQWater.

Non-Reducing Solution:

Add 1 μl, of MilliQ water to a sample buffer aliquot (25 μL) and vortexfor 5 seconds. Use the non-reducing solution within its preparation day.

Reducing Solution:

Add 1 μl, of the according DTf-Solution to a sample buffer aliquot (25μL) and vortex for 5 seconds. Use the reducing solution within itspreparation day.

Sample Preparation:

-   -   Samples are analysed at the concentration ranging between 2.4-3        mg/ml.    -   If it's necessary the samples can be diluted to the target        concentration using Milli Q water.

Samples are prepared according to the Reagent Kit Guide using thereducing and non-reducing sample buffers according to the instruction inthe Reagent Kit Guide and also mentioned above. It is stronglyrecommended to use, differently from the guide, greater volumes toachieve reproducible and accurate results. An example how to prepare theladder and the samples is reported below:

Sample Preparation Solution Reducing and Non Reducing Condition

Volume Total Volume Reagent μL μL Sample diluted at 3 mg/mL 3 μL  6 μLSample buffer (reducing or 2 μL not reducing) Maleimide solution 1 μLSamples have to be mixed (via vortex) well and spun down All samples andthe Ladder are heated 5 minutes at 70° C. MilliQ water 84 μL  90 μLVortex well and spin down Loading 6 μL all Sample and Ladder

Note 1: For the IPCs whose concentration is between 2.4 mg/ml and 3.0mg/mL, the sample preparation follows the table above but the volume ofMilliQ Water added after sample heating is calculated in order to reacha final protein concentration of 0.1 mg/ml.

An example of sample preparation for a sample having concentrationbetween 2.4 and 3.0 mg/mL, is reported here below:

Volume Total Volume Reagent μL μl Sample (2.6 mg/mL) 3 μL  6 μL Samplebuffer (reducing or 2 μL not reducing) Maleimide solution 1 μL Sampleshave to be mixed (via vortex) well and spundown All samples and theLadder are heated 5 minutes at 70° C. MilliQ water 72 μL  78 μL vortexwell and spin down Loading 6 μL all Sample and Ladder

Note 2: All wells have to be loaded. If the sample number is lower thanthe available wells, the empty wells can be used for additionalduplicates or blank samples.

Preparing the System and the Chip:

-   -   To clean the system before and as well after an analysis fill        the “Electrode Cleaner” with 600IIL MilliQ Water and place it        into the Agilent 2100 Bioanalyzer, close the lid and let the        system cease. No further action is required.    -   Adjust the base-plate of the chip priming station to position        “A” and the syringe clip to its middle position.        Preparing the Chip

System Preparation Insert a new Protein Chip in the priming stationPipette 12 μL of Gel-Dye Mix in the well marked G (Up right) Set theplunger at 1 mL and close the chip priming station Press the plungeruntil it is held by the clip Wait 60 seconds and then release the clipWait 5 seconds and slowly pull back the plunger to the 1 mL mark Openthe chip priming station Remove the solution in this well Pipette 12 μLof Gel-Dye Mix in the well marked G (Up right) and in all remainingwells marked with G Pipette 12 μL of Destaining Solution in the wellmarked with DSLoading of the Ladder and the Sample:

-   -   Transfer 6 μL of each sample into a sample well and as well 6 μL        of the ladder in the dedicated well, which is clearly indicated        with a ladder symbol.

Place the chip into the Agilent 2100 Bioanalyzer and start the analysiswithin 5 min.

Example of Sample Set

Amount Well Sample μL 1 Blank 6 2 Blank 6 3 Unknown sample1 rep1 6 4Unknown sample1 rep2 6 5 Unknown sample2 rep1 6 6 Unknown sample2 rep2 67 Unknown sample3 rep1 6 8 Unknown sample3 rep2 6 9 Current ReferenceMaterial Rep 1 6 10 Current Reference Material Rep 1 6 Ladder Ladder 6Data Analysis and Evaluation of the Results:

To gain results the following minimum operations have to be executed

-   -   Place the chip in the specific spot and close the lid.    -   In the instrument context select        Assay—Electrophoresis-Protein-Protein 230 Plus.    -   Click on START to start the analysis, which is completed within        30 minutes.    -   The raw data are shown by clicking on “Data Analysis” where all        experiments, carried out at the day, are listed. Click on the        experiment of interest and select it.    -   The gel generated from the chosen experiment is automatically        opened.    -   Data can be shown as an electropherogram or gel-like image.

Detailed information regarding the integration of the peaks in theelectropherogram (to gain the purity data) is included in the manual ofthe software. The purity of a sample is automatically given by thesystem by automatic integration, but if needed, the manual integrationcan be applied.

Results:

In non-reducing condition the results are indicated as % Purity, and %LMW (sum of peaks before monomer).

In reducing condition the results are indicated as % Purity, as sum ofheavy chain and light chain.

The indicative molecular weight values are reported in the table below:

Indicative Molecular Weight of Adalimumab

Condition Results KDa Non Reducing Monomer 151 Reducing LC 27 HC 582. Unfolding Temperature—DSF

DSF (differential scanning fluorimetry) was performed as follows:

2 microliters of Sypro Orange (Orange protein gel stain, cod. S6650,Life Technologies) previously diluted 500-fold in water for injectionwere added to 20 microliters of drug product solution. Upon addition ofSypro Orange, the DP solutions (triplicate preparation) are filled in96-well plates (MicroAmp Fast 96-W Reaction Plate 0.1 mL, cod. 4346907).The plates are then sealed with a protective, transparent cover(MicroAmp Optical Adhesive Film, cod. 4311971) and then subjected tocentrifugation to remove air bubbles. The plates are then inserted inthe 7500 Fast Real-Time AB Applied Biosystem PCR system and scanned foremission profiles at temperatures from room temperature to 90-100° C.The dependence of intensity of fluorescence emission on temperature is acurve which typically shows an inversion point/discontinuation at thedenaturation temperature, parameter used to compared the differentcompositions.

3. Isoforms Profile—iCE280

cIEF by iCE280 (isoforms profile): After purification and removal ofsalts with centrifugation in Amicon Ultra-4 centrifugal devices (cut off10 kDa), the samples were pre-diluted to the concentration of 5.0 mg/mLwith purified water. A second dilution was then made to 1.0 mg/mL with asolution composed of: methyl cellulose, Pharmalyte 5-8 (GE Healthcare),Pharmalyte 8-10.5 (GE Healthcare), low pI marker 7.05 (Protein Simple),high pI marker 9.50 (Protein Simple) and purified water. Upon dilutionthe samples were centrifuged at 10000 rpm for 3 minutes. An additionalcentrifugation step (2 minutes at 7000 rpm) is then conducted on 150microL of each sample transferred in glass inserts. The cIEF (capillaryisoelectric focusing) was carried out with the iCE280 system by ProteinSimple, using capillary cartridges Fc with 100 micron ID coating andtotal length of 50 nm (Cat. No. 101700/101701 by Protein Simple). Theseparation of the various isoforms is made using 100 mM sodium hydroxide(in 0.1% methyl cellulose) as a cathodic solution and 80 mM o-phosphoricacid (in 0.1% methyl cellulose) as an anodic solution. Theelectropherogram is acquired at 280 nm over pre-focusing and focusingtimes of 1 and 6 minutes respectively, at a voltage of 1500 V(pre-focusing) and 3000 V (focusing).

4. Protein Content—OD

OD (protein content) measurements were taken on samples which wereinitially diluted gravimetrically (triplicate independent dilutions weremade) with relevant buffer or placebo from starting concentration toabout 10 mg/mL. The diluted solutions were tested for absorbance at 280and 320 nm in 0.1 cm pathlength quartz cuvettes, at room temperature,with a double-beam spectrophotometer (Lambda35 by Perkin Elmer). Thevalue of 1.35 was used as molar extinction coefficient of Adalimumab.

5. Aggregates Determination—SE-HPLC

The samples were diluted with DPBS 1× to a concentration of 0.5 mL andinjected (20 microL injection volume) in a Column TSK gel Super SW30004.6 mm ID×30.0 cm cod.18675 by Tosoh maintaining isocratic conditions(mobile phase: 50 mM Sodium Phosphate+0.4M Sodium perchlorate, pH6.3±0.1). UV detection was made at 214 nm at a flow rate of 0.35 mL. Theduration of each analytical run was 15 minutes. Prior to the analysisthe samples were maintained at 2-8° C. in the autosampler of the WatersAlliance HPLC systems used for this test.

6. Turbidity—Nephelometry

Turbidity was assessed via nephelometric (effect based on the lightdiffusion effect caused by particles with dimensions typically <1micron) measurements conducted with a turbidimeter 2100AN ISTurbidimeter by Hach at room temperature. Minimum amounts of 3 mL ofsolution were placed in reduced volume glass cuvettes and tested fordiffusive effect after prior calibration of the instrument with a seriesof standard solutions (0.1-7500 NTU).

7. Osmolality Determination—Osmolality

Osmolality was measured based on the cryoscopic characteristic of thesolutions. The tests were conducted with an Osmomat 030-D by Gonotechsubjecting 50 microL of the sample to freezing. The freezing temperaturedepends on the osmolality of the solution (i.e. on the presence ofagents dissolved such as salts, sugars, other ionic and non-ionicspecies, etc).

8. pH Determination—pH

pH was determined using potentiometric measurements conducted at roomtemperature with Mettler Toledo Seven Multi pH meters.

9. Particle Count—Sub-Visible Particles

The samples were 5-fold diluted with purified water to a final volume of25 mL. The number of particles are determined at room temperature byPAMAS SVSS by Aminstruments collecting four independent runs andaveraging the results for each respective dimensional fraction ofinterest.

Example 1—Formulations for First Formulation Screen

The following first set of formulations (often referenced herein as DoE1formulations) are shown below in Table 1.

TABLE 1 List of DoE1 formulations for later Screen Experiments 1 Salt(NaCl) concen- Buffer Form tration type # (mM) (10 mM) pH Stabilizer 125 Acetate 4.6 Lysine Hydrochloride (100 mM) 2 100 Acetate 4.6 ArginineMonohydrocloride + Aspartic Acid (80 mM + 20 mM) 3 50 Acetate 4.8Arginine Monohydrocloride + Aspartic Acid (80 mM + 20 mM) 4 25 Acetate4.8 Mannitol (200 mM) 5 75 Acetate 4.8 Mannitol (200 mM) 6 50 Acetate5.0 Lysine Hydrochloride (100 mM) 7 100 Acetate 5.0 Trehalose dihydrate(200 mM) 8 25 Acetate 5.2 Trehalose dihydrate (200 mM) 9 75 Acetate 5.2Arginine Monohydrocloride + Aspartic Acid (80 mM + 20 mM) 10 75 Acetate5.2 Trehalose dihydrate (200 mM)

The formulations of Table 1 were manufactured starting from apreformulated, surfactant-free DS material.

An aliquot of the DS was diafiltrated with 10 mM sodium acetate/aceticacid buffer at pH 5.0 until a three-fold volume exchange with the bufferwas achieved. Then the required excipients have been added to thebuffer-exchanged DS materials and the pH adjusted to the target byaddition of a diluted solution of sodium hydroxide. Each formulation wasfiltered through 0.22 μm PES filters.

In Table 2, the results in terms of material recovery and osmolality forthe three buffer-exchanges DS materials are reported.

TABLE 2 Recovery and osmolality of the DS materials after bufferexchange After exchange Starting DS Starting DS Protein Final FinalProtein volume Concentration Treated Volume Concentration RecoveredRecovery Osmolality Buffer (mL) (mg/mL) (mg) (mL) (mg/mL) (mg) (%)(mOsm/kg) Acetate 200 63.3 12660 180 65.2 11736 93 30

There was good recovery for the sodium acetate buffer (>90%). Theosmolality values indicate the satisfactory degree of buffer exchangereached, with a minimal residual of species coming from the originatingDS.

Example 2—Formulations for Second Formulation Screen

The following second set of formulations (often referenced herein asDoE2 formulations) are shown below in Table 3 (as derived from Table 4below that).

TABLE 1 List of DoE2 formulations for later Screen Experiments 2(formulations derived from that presented in Table 4 with the extrasurfactant indicated) Polysorbate 80 concentration (mg/mL) Formulations0 0.5 1 Form 1 (deriving from Form A, x — — Table 4) Form 2 (derivingfrom Form A, — x — Table 4) Form 3 (deriving from Form A, — — X Table 4)

TABLE 4 Formulation prototype deriving from the DoE1 screen Salt Buffer(NaCl) type Form mM (10 mM) pH Stabilizer A 75 Acetate 5.2 Trehalosedihydrate (200 mM)

The DoE2 formulations (Table 3) were manufactured starting from apreformulated, surfactant-free, DS material.

As before, aliquots of the DS have been diafiltrated until a three-foldvolume exchange was achieved. Then the required excipients have beenadded to the buffer-exchanged DS materials and the pH adjusted to thetarget by addition of a diluted solution of sodium hydroxide. Eachformulation was filtered through 0.22 μm PES filters.

In Table 5, the results in terms of osmolality and turbidity for thebuffer-exchanges DS materials are reported.

The osmolality values (≤40 mOsm/kg) indicated the satisfactory degree ofbuffer exchange reached, with a minimal residual of species coming fromthe originating DS.

TABLE 5 Osmolality and turbidity of the DS materials after bufferexchange Turbidity Osmolality Buffer (NTU) (mOsm/kg) Acetate 19 29

Example 3—Comparative Formulations for Both First and Second Screens

For comparison and control purposes, three reference formulations wereprepared or obtained, including Ref-1 (Humira® composition manufacturedby the Applicant); Ref-2 (RMP US—Humira® commercial drug product fromthe USA); and Ref-3 (RMP EU—Humira® commercial drug product from theEU). All of these reference formulations had the composition shown inTable 6.

TABLE 6 Composition of Humira DP Amount per container (mg) Ingredient(filling volume = 0.8 mL) Amount (mg/mL) Adalimumab 40 50 Citric Acid1.04 1.3 Monohydrate Dibasicsodium 1.22 1.53 phosphate dihydrateMannitol 9.6 12 Monobasicsodium 0.69 0.86 phosphate dihydratePolysorbate 80 0.8 1 Sodium chloride 4.93 6.16 Sodium citrate 0.24 0.3WFI and sodium q.b. to adjust pH to 5.2 q.b. to adjust pH to 5.2hydroxideScreening

A first formulation screen (DoE1) led to the identification of variousfactors (e.g. pH, presence of NaCl, excipient type) responsible forprotein stability, and ultimately to the selection of formulations to bepursued in a second screen (DoE2), which sought to fine-tune theformulations and evaluate how surfactants, such as Polysorbate 80, canimpact the stability of the protein.

Each of the two screens involved various analytical testing, as definedhereinbefore and referred to hereinafter, upon a range of differentformulations which were exposed to varying levels of thermal,mechanical, and light stress over prolonged periods (e.g. 1 month).These formulation screens enabled the gathering of a significant amountof data, which provided surprising and valuable insights allowing forthe development of new advantageous formulations.

The results of the two formulations screens are presented below.

Screening Experiment 1—Analysis and Screening of Example 1 FormulationsAgainst Comparative Formulations of Example 3

Preliminary DoE screening (Step 1) evaluated the effect that ionicstrength (given by NaCl), pH and different stabilizers exerts on theprotein in the course of short term stability studies.

A response surface D-Optimal statistical design has been applied. Threefactors were considered:

-   -   Ionic strength (driven by NaCl concentration, which was varied        in the range 25 mM-100 mM and was set as a numeric factor);    -   pH (the range 4.6-6.4 buffered by acetate, was investigated;    -   Stabilizer/Excipient (categoric factor comprising several        levels: Lysine Hydrochloride, Arginine+Aspartic Acid, Mannitol,        Trehalose Dihydrate).

These formulations were manufactured, as described in Example 1 above,starting from DS without Polysorbate 80 and were thereforesurfactant-free.

Table 7 below summarizes the formulations tested within this screening.In addition to the 10 formulations proposed, two controls have also beenanalysed as comparators:

-   -   Humira commercial drug product “DP” (Formulated as per Example 3        above)    -   MS drug substance “DS” formulated as Humira commercial DP        (Formulated as per Example 3 above)

TABLE 7 List of DoE1 formulations (Step 1) screened through thermalstress conditions (stability at 40° C.) and high throughputdetermination of protein unfolding temperature (DSF). Salt (NaCl) BufferForm conc type # (mM) (10 mM) pH Stabilizer 1 25 Acetate 4.6 LysineHydrochloride (100 mM) 2 100 Acetate 4.6 Arginine Monohydrocloride +Aspartic Acid (80 mM + 20 mM) 3 50 Acetate 4.8 ArginineMonohydrocloride + Aspartic Acid (80 mM + 20 mM) 4 25 Acetate 4.8Mannitol (200 mM) 5 75 Acetate 4.8 Mannitol (200 mM) 6 50 Acetate 5.0Lysine Hydrochloride (100 mM) 7 100 Acetate 5.0 Trehalose dihydrate (200mM) 8 25 Acetate 5.2 Trehalose dihydrate (200 mM) 9 75 Acetate 5.2Arginine Monohydrocloride + Aspartic Acid (80 mM + 20 mM) 10 75 Acetate5.2 Trehalose dihydrate (200 mM) Ref-1 Humira composition (formulationmanufactured with MS (MS) Drug Substance) - Example 3 Ref-2 Humiracommercial DP (USA) - Example 3 (RMP US) Ref-3 Humira commercial DP(EU) - Example 3 (RMP EU)

The formulations were tested according to the plan reported in Table 8.Thermal stress up to 1 month at 40° C. was considered. High throughputassessment made with the DSF technique (aimed at a fast screening basedon determination of protein unfolding temperature) was performed at TO.

TABLE 8 Panel of analytical tests carried out on preliminary DoEformulations (Step 1): 1-month thermal stress conditions at 40° C.Accelerated (40° C.) Stability time (weeks) Methods Test 0 2 w 4 w ODContent x — x SE-HPLC Aggregates x x x Bionalyzer Purity x x x pH pH x xx Osmolality Osmolality x — — DSF UnfoldingT x — —1.1 Osmolality Screen

The osmolality of the DoE1 formulations compounded starting from thebuffer exchanges DS materials (par. 5.1.1) is reported in Table 9.

Most formulations were found in the range of osmolality of 250-400mOsm/kg, while slightly higher values were observed at the highestsodium chloride concentrations.

TABLE 9 Osmolality (mOsm/kg) recorded at time 0 for DoE1 screeningformulations Salt (NaCl) Buffer concen- type Form tration (10 Time #(mM) mM) pH Stabilizer 0 1 25 Acetate 4.6 Lysine Hydrochloride (100 mM)0.302 2 100 Acetate 4.6 Arginine Monohydrocloride + 0.407 Aspartic Acid(80 mM + 20 mM) 3 50 Acetate 4.8 Arginine Monohydrocloride + 0.339Aspartic Acid (80 mM + 20 mM) 4 25 Acetate 4.8 Mannitol (200 mM) 0.322 575 Acetate 4.8 Mannitol (200 mM) 0.423 6 50 Acetate 5.0 LysineHydrochloride (100 mM) 0.342 7 100 Acetate 5.0 Trehalose dihydrate (200mM) 0.506 8 25 Acetate 5.2 Trehalose dihydrate (200 mM) 0.341 9 75Acetate 5.2 Arginine Monohydrocloride + 0.366 Aspartic Acid (80 mM + 20mM) 10 75 Acetate 5.2 Trehalose dihydrate (200 mM) 0.441 ReferenceIn-House 0.374 (Humira composition, Merck Serono DS) RMP (USA) Humira NARMP (EU) Humira 0.3101.2 Protein Content (OD)

The protein content of the DoE1 formulations was determined at time 0and after 1 month at 40° C.

FIG. 1 is a bar chart showing the protein content (mg/mL) of the DoE1formulations (of Example 1), along with reference standards(representing comparator HUMIRA® formulations), at an arbitrary startpoint (blue bars, time=0) and after 4 weeks (red bars) of theformulation(s) being heated at 40° C.

The results presented in FIG. 1, indicated no significant changesoccurring over time. All concentrations were found in line with thetarget of 50 mg/mL.

1.3 Aggregation (SE-HPLC)

FIG. 2 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE1 formulations (of Example 1), along with referencestandards (representing comparator HUMIRA® formulations), at anarbitrary start point (blue bars, time=0) and after both 2 weeks (greenbars) and 4 weeks (orange bars) of the formulation(s) being heated at40° C. The total aggregates observed by SE-HPLC over stability at 40° C.are graphically represented in FIG. 2. Minimal increases in aggregationwere observed in all formulation. However, even after 1 month, allaggregation levels amounted to less than 1%.

1.4 Fragmentation (Bioanalyzer)

FIG. 3 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE1 formulations (of Example 1), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (dark blue bars, time=0) and after both 2 weeks(pink bars) and 4 weeks (light blue bars) of the formulation(s) beingheated at 40° C.

In FIG. 3, the variation of fragments over time as determined byBioanalyzer is reported. Formulations at more acidic pH (typically lowerthan or equal to 5.0, as in Formulations 1-7) tend to undergo fasterfragmentation rates. Moreover, the presence of amino acids at this pHrange can considerably worsen the stability profile (Formulations1-2-3). The detrimental effect of aminoacidic species is also confirmedat pH 5.2-5.4 (Formulation 9).

At pH>5.0 and in presence of sugar/polyols, all the formulas, includingthe references, are comparable (fragmentation lower than 1% after 1month at 40° C.).

The data were analysed by means of ANOVA which confirmed the statisticalsignificance of the pH factor (p-value<0.001), also indicating that pHvalues>5.0 should be targeted to minimize fragmentation.

Sodium chloride was not found to be a factor critical to stability inthe range 25-100 mM.

1.5 pH Screening

Table 10 shows the pH of the DoE1 formulations (of Example 1), alongwith reference standards (representing comparator HUMIRA® formulations),at an arbitrary start point (time=0) and after both 2 weeks and 4 weeksof the formulation(s) being heated at 40° C.

As can be seen from Table 10, no deviations from targeted pH wereobserved.

TABLE 10 pH of DoE1 screening formulations determined over stability at40° C. Salt (NaCl) Buffer Stability time Form conc type Time 2 weeks 4weeks # (mM) (10 mM) pH Stabilizer 0 40° C. 40° C. 1 25 Acetate 4.6Lysine Hydrochloride (100 mM) 4.6 4.7 4.7 2 100 Acetate 4.6 ArginineMonohydrocloride + 4.7 4.6 4.7 Aspartic Acid (80 mM + 20 mM) 3 50Acetate 4.8 Arginine Monohydrocloride + 4.8 4.8 4.8 Aspartic Acid (80mM + 20 mM) 4 25 Acetate 4.8 Mannitol (200 mM) 4.8 4.8 4.8 5 75 Acetate4.8 Mannitol (200 mM) 4.8 4.8 4.8 6 50 Acetate 5.0 Lysine Hydrochloride(100 mM) 5.0 5.0 5.0 7 100 Acetate 5.0 Trehalose dihydrate (200 mM) 5.05.0 5.0 8 25 Acetate 5.2 Trehalose dihydrate (200 mM) 5.2 5.2 5.2 9 75Acetate 5.2 Arginine Monohydrocloride + 5.1 5.2 5.2 Aspartic Acid (80mM + 20 mM) 10 75 Acetate 5.2 Trehalose dihydrate (200 mM) 5.2 5.2 5.2Reference In-House (Humira composition, Merck Serono DS) 5.2 5.2 5.2 RMP(USA) Humira 5.3 5.3 5.3 RMP (EU) Humira 5.3 5.3 5.31.6 Unfolding Temperature (DSF)

DSF is a high throughput method which aims at the determination of theunfolding temperature of proteins by virtue of increasing interactionswith fluorescent probes as temperature ramps are applied to the samples.When the protein starts to unfold, it will progressively exposehydrophobic patches to the solvent attracting the fluorescent probesthat will pass from the free state in solution (non-fluorescent) to thebound state (via hydrophobic interactions) with the protein, thusincreasing the degree of fluorescent signal.

From the evaluation of the fluorescence signal, it was possible todetermine the midpoint of the sigmoidal curves, which indicates thetransition point of each formulation. It is assumed that the higher thetransition point, the higher the resistance of the formula to thermalstress.

The results of the assessment conducted on the DoE1 screeningformulations are reported in FIG. 4. FIG. 4 is a bar chart showing theunfolding temperature (° C.), as determined by DSF, of the DoE1formulations (of Example 1), along with reference standards(representing comparator HUMIRA® formulations).

The unfolding temperature of the three reference formulations is 71-72°C. Few formulations, aside from the references, were found to haveunfolding temperatures higher than 70° C., but those that did include:

-   -   Formulations 8 and 10 (formulations in sodium acetate buffer pH        5.2+Trehalose dihydrate at varying sodium chloride        concentrations)

Therefore, this test confirmed the results previously obtained forfragmentation by Bioanalyzer: polyols/sugars can positively impact thethermal stability of the protein, especially at pH>5.0, while sodiumchloride does not seem to significantly affect its behavior.

The ANOVA model for response surface identified pH (p-value<0.01) andexcipient (0.01<p-value<0.05) as the significant factors affectingprotein stability.

1.7 Iso Forms Profile Change Vs RMP

The isoforms profile of DoE screening formula 10 has been tested after10-11 weeks at 40° C. and compared to Reference samples.

The data, in terms of main peak and acidic cluster variations, arereported in Table 11.

Comparable variations are obtained for the five samples tested.

TABLE 11 Isoforms profile by iCE280 of most promising formulations fromDoE screening 1 and references. ID Time 0 10 weeks (40° C.) 11 weeks(40° C.) Main DoE1-10 56.7 40.1 — Ref-1 (MS) 55.8 38.5 — Ref-3 RMP (EU)56.5 40.7 — Ref-2 RMP (US) 56.8 40.6 — Acidic cluster DoE1-10 19.3 39.3— Ref-1 (MS) 19.8 40.5 — Ref-3 RMP (EU) 19.5 38.9 — Ref-2 RMP (US) 20.239.8 —Conclusion of Screening Experiment 1

The results obtained from Bioanalyzer and DSF testing were combinatelyevaluated by means of the ANOVA model for response surfaces in order todetermine the best compositions that can possibly guarantee the highestthermal stability to the protein.

The list of the compositions recommended are reported in Table 12, whichalso compares the performances of the resulting prototypes formulationswith the Humira RMP, in terms of unfolding temperature and fragmentationchange over 1 month at 40° C.

Formulation A corresponds to DoE1 Formulation 10 and the real data werereported.

Comparing these formulas to the RMP it can be concluded that thebehavior of these prototype formulations in response to thermal stressis comparable with that observed for the RMP.

TABLE 12 Outcome of DoE1 experiments: recommended compositions forsecond screen Salt Buffer (NaCl) type Form mM (10 mM) pH Stabilizer A 75Acetate 5.2 Trehalose dihydrate (200 mM)

Somewhat unexpectedly, formulations containing trehalose dihydrate asthe sole stabilizer performed extremely well, especially in terms offragmentation inhibition, unfolding inhibition, and pH maintenance. Suchtrehalose-based formulations also exhibited good performance in terms ofaggregation and precipitation. That trehalose was such a strongcandidate as a stabiliser, especially on its own, was extremelypromising in view of its antioxidant properties, which would impartfurther long-term chemical stability (especially vis a vis oxidationand/or photo-oxidation) to adalimumab formulations. Furthermore, thattrehalose can be used alone and yet still exhibit excellent performance,was considered especially encouraging and paved the way to less complexformulations employing fewer components—this would in turn reduceprocessing and costs associated with the production of the relevantadalimumab drug product. As such, these trehalose-based formulationswere taken into a second round of screening experiments in order tofine-tune the formulations.

Screening Experiment 2—Analysis and Screening of Example 2 FormulationsAgainst Comparative Formulations of Example 3

A formulation prototype from the previous screen was identified (Table12). Since the previous step was conducted with no surfactant added, thesecond step aimed to screen a series of levels of compounded Polysorbate80 surfactant (range: 0-1 mg/mL) in order to assess whether surfactantaddition is required to favor protein stability.

Table 3 (Example 2) summarizes the design of this second step of thestudy and lists the formulations (DoE2 formulations) tested in thissecond screening exercise.

Typically, surfactants have been observed to contrast mechanicalstress-induced aggregation and shaking stress tests have been thereforeexecuted so as to evaluate how Polysorbate 80 affects protein stabilityand response to shaking.

As with Step 1, the reference compositions described in Example 3 havealso been evaluated so as to provide a baseline for the development of anew formulation.

The complete list of analyses conducted on this block of formulations isreported in Table 13. In this second screen, the respective formulationswere exposed to three different types of stress, thermal, mechanical,and light.

TABLE 13 Panel of analytical tests carried out on DoE2 formulations(Step 2): 1-month thermal stress conditions at 40° C. (A), shakingstress at 200 rpm (B) and light exposure according to ICH Q1B (C). A.Thermal stress at 40° C. Accelerated (40° C.) Stability time (weeks)Methods Test 0 2 w 4 w OD Content x — x iCE280 Isoforms x x x SE-HPLCAggregates x x x Bionalyzer Purity x x x pH pH x x x OsmolalityOsmolality x — — Nephelometry Turbidity x x x DSF UnfoldingT x — — B.Shaking stress conditions Shaking stress (200 rpm) Stability time(hours) Methods Test 0 24 h 48 h OD Content x — — SE-HPLC Aggregates x xx Bioanalyzer Purity x x x pH pH x x x Nephelometry Turbidity x x x C.Light Exposure 7 hours of exposure at 765 W/m² (ICH Q1B). Light exposureSample Methods Test Time 0 Exposed OD Content x — iCE280 Isoforms x xSE-HPLC Aggregates x x Bioanalyzer Purity x x pH pH x x NephelometryTurbidity x x

Thermal stress tests were performed by simply heating a sample of therelevant formulations at the stipulated temperature for the stipulatedamount of time (typically 2 weeks or 4 weeks/1 month).

Mechanical stress tests were performed by simply mechanically shaking asample of the relevant formulations at room temperature at 200 rpm forthe stipulated period of time (typically 24 hours or 48 hours).

Light stress tests were performed by simply exposing a sample of therelevant formulations to 765 W/m² light (in accordance with ICH Q1Bguidelines of the European Medicines Agency in relation tophotostability testing of new active substances and medicinal products)for 7 hours.

2.1 Osmolality

The osmolality of the DoE2 screening formulations are reported in Table14. The values, comprised in the range 378-401 mOsm/kg are probablyoverestimated due to the presence of Trehalose dihydrate that can leadto some increase in viscosity affecting the cryoscopic point of thesolutions and hence the osmolality. This was confirmed by measurementsin relation to other test formulations, which were 3-fold diluted withWFI prior to the osmolality test in order to decrease the viscosity: thereal osmolality of all these formulas is <350 mOsm/kg.

TABLE 14 Osmolality of DoE2 screening formulations (tested undiluted)Salt (NaCl) Surfactant concen- Buffer (Polysorbate 80) Form tration typeconcentration Time # (mM) (10 mM) pH Stabilizer (mg/mL) 0 DoE2-l 50Acetate 5.2 Trehalose 0 390 dihydrate (200 mM) DoE2-2 50 Acetate 5.2Trehalose 0.5 386 dihydrate (200 mM) DoE2-3 50 Acetate 5.2 Trehalose 1388 dihydrate (200 mM)2.2 Protein Content (OD)

The protein content of all the DoE2 formulations at time 0 were in linewith the protein concentration target of 50 mg/mL (Table 15).

TABLE 15 Protein content (OD) of DoE2 screening formulations (testedundiluted) Salt (NaCl) Surfactant concen- Buffer (Polysorbate 80) Formtration type concentration Time # (mM) (10 mM) pH Stabilizer (mg/mL) 0DoE2-1 50 Acetate 5.2 Trehalose 0 50.6 dihydrate (200 mM) DoE2-2 50Acetate 5.2 Trehalose 0.5 52.0 dihydrate (200 mM) DoE2-3 50 Acetate 5.2Trehalose 1 51.3 dihydrate (200 mM)2.3 Aggregates with Thermal Stress (SE-HPLC)

The variations in total aggregates by SE-HPLC are presented in FIG. 5 isa bar chart showing the % aggregation, as determined by SE-HPLC, of theDoE2 formulations (of Example 2), along with reference standards(representing comparator HUMIRA® formulations), at an arbitrary startpoint (red bars, time=0) and after both 2 weeks (green bars) and 4 weeks(purple bars) of the formulation(s) being heated at 40° C.

Minimal changes were observed for all the formulation, being the totalaggregates amount after 1 month at 40° C. below 1%.

The performances of the DoE2 screening formulations arecomparable/slightly better than those of the RMP materials.

2.4 Fragmentation with Thermal Stress (Bioanalyzer)

The variations in fragments by Bioanalyzer are presented in FIG. 6. FIG.6 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations), atan arbitrary start point (blue bars, time=0) and after both 2 weeks (redbars) and 4 weeks (green bars) of the formulation(s) being heated at 40°C.

In sodium acetate the presence of Polysorbate 80 at 1 mg/mL seems toslightly improve stability (cfr. DoE2-3 vs. DoE2-1 and DoE2-2) providingperformances comparable to those observed in the RMP.

2.5 Isoforms Profile with Thermal Stress (iCE280)

The main peak and acidic cluster changes of the three formulations over1 month at 40° C. are reported in FIGS. 7 and 8 respectively

FIG. 7 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)at an arbitrary start point (blue bars, time=0) and after both 2 weeks(red bars) and 4 weeks (green bars) of the formulation(s) being heatedat 40° C.

FIG. 8 is a bar chart showing the acid cluster peak(s) isoforms profile,as determined by iCE280 analysis, of the DoE2 formulations (of Example2) at an arbitrary start point (blue bars, time=0) and after both 2weeks (red bars) and 4 weeks (green bars) of the formulation(s) beingheated at 40° C.

These results confirm the experimental evidences already highlighted byiCE280 on the prototype formulations (resulting from the firstscreening).

The results, in terms of acidic cluster, are in line with theobservations made for the main peak.

2.6 pH Screen with Thermal Stress

The variation in pH of the DoE2 formulations (of Example 2) over aperiod of time during which the formulations are heated at 40° C. isshown in Table 16.

pH was entirely stable over the testing period.

TABLE 16 DoE2 screening: pH (thermal stress at 40° C.) Salt Surfactant(NaCl) Buffer (Polysorbate 80) Form concentration type concentrationTime 2 weeks 4 weeks # (mM) (10 mM) pH Stabilizer (mg/mL) 0 (40° C.)(40° C.) DoE2-1 50 Acetate 5.2 Trehalose 0 5.2 5.2 5.2 dihydrate (200mM) DoE2-2 50 Acetate 5.2 Trehalose 0.5 5.2 5.3 5.3 dihydrate (200 mM)DoE2-3 50 Acetate 5.2 Trehalose 1 5.2 5.3 5.3 dihydrate (200 mM)2.7 Turbidity with Thermal Stress (Nephelometry)

FIG. 9 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 2 weeks (red bars) and 4weeks (green bars) of the formulation(s) being heated at 40° C.

The turbidity of the three formulations is, time 0, in the range oftypically opalescent solutions (6-18 NTU). With respect to theoriginating DS materials, of typical turbidity of 19-52 NTU, the DPsolutions after aseptic filtration are considerably clarified.

Importantly, turbidity values of Humira RMP are normally around 10 NTU,in line with our form ulas.

2.8 Aggregates with Mechanical Stress (SE-HPLC)

FIG. 10 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2) at an arbitrary startpoint (blue bars, time=0) and after both 24 hours (red bars) and 48hours (green bars) of the formulation(s) being mechanically agitated(shaking).

The variations in total aggregates by SE-HPLC are presented in FIG. 10.

Remarkably, no change (DoE2-1 and DoE2-3) or a maximum change of +0.1%(DoE2-2) are observed.

2.9 Fragmentation with Mechanical Stress (Bioanalyzer)

FIG. 11 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking).

The variations in fragments by Bioanalyzer are presented in FIG. 11.Minimal changes are observed, being all the values recorded equal to orlower than 0.5%.

After 48 hour shaking at room temperature all the samples presentedfragmentation in the range 0.2-0.4%. No trend towards fragmentationincreases was highlighted upon mechanical shaking.

2.10 pH Screening with Mechanical Stress

The variation in pH of the DoE2 formulations (of Example 2) over aperiod of time during which the formulations are mechanically agitated(shaking) is shown in Table 17. No changes where observed.

TABLE 17 DoE2 screening: pH (mechanical shaking) Salt Surfactant (NaCl)Buffer (Polysorbate 80) Form concentration type concentration Time 24 48# (mM) (10 mM) pH Stabilizer (mg/mL) 0 hours hours DoE2-1 50 Acetate 5.2Trehalose 0 5.2 5.2 5.2 dihydrate (200 mM) DoE2-2 50 Acetate 5.2Trehalose 0.5 5.2 5.3 5.3 dihydrate (200 mM) DoE2-3 50 Acetate 5.2Trehalose 1 5.2 5.3 5.3 dihydrate (200 mM)2.11 Turbidity with Mechanical Stress (Nephelometry)

FIG. 12 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) at an arbitrarystart point (blue bars, time=0) and after both 24 hours (red bars) and48 hours (green bars) of the formulation(s) being mechanically agitated(shaking). No changes were observed.

2.12 Aggregates with Light Stress (SE-HPLC)

FIG. 13 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations), beforeexposure to light (blue bars, time=0) and after 7-hour light exposure at765 W/m² (red bars).

Comparisons were also made with Humira samples (from US and EU) treatedat the same conditions. In the RMP, aggregation increases up to 9-15%upon light exposure (at time 0 aggregates are lower than 1%). All theDoE2 formulations present lower or comparable increases and thereforebetter/similar resistance to thermal stress. More in detail:

-   -   Formulations in acetate buffer: 4.3-6.8% total aggregates upon        light exposure        2.13 Fragmentation with Light Stress (Bioanalyzer)

FIG. 14 is a bar chart showing the % fragmentation, as determined by aBioanalyzer, of the DoE2 formulations (of Example 2), along withreference standards (representing comparator HUMIRA® formulations),before exposure to light (blue bars, time=0) and after 7-hour lightexposure at 765 W/m² (red bars).

Minimal increases were highlighted (+0.3% at most, after exposure). Allfragmentation amounts are well below 1% after 7-hour exposure (FIG. 14).

2.14 Isoforms Profile with Light Stress (iCE2280)

FIG. 15 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2),along with reference standards (representing comparator HUMIRA®formulations), before exposure to light (blue bars, time=0) and after7-hour light exposure at 765 W/m² (red bars).

FIG. 16 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2), along with reference standards (representing comparatorHUMIRA® formulations), before exposure to light (blue bars, time=0) andafter 7-hour light exposure at 765 W/m² (red bars).

In the Humira RMP, the light exposure determines significant effects:most relevantly, decreases in main peak abundance (around −9%) andconcurrent increase in acidic cluster (up to +15%), related tophotooxidation phenomena, are observed.

Acetate formulas can considerably improve the resistance of the proteinto degradation phenomena: decreases in main peak abundance are around−3.5% or lower, increases in acidic cluster scored a maximum of +4%.

2.15 Turbidity with Light Stress (Nephelometry)

FIG. 17 is a bar chart showing the turbidity, as determined byNephelometry, of the DoE2 formulations (of Example 2) before exposure tolight (blue bars, time=0) and after 7-hour light exposure at 765 W/m²(red bars). Essentially no changes were observed.

2.16 pH Screen with Light Stress

The variation in pH of the DoE2 formulations (of Example 2), over aperiod of time during which the formulations are exposed for 7-hours tolight at 765 W/m², is shown in Table 18. No changes where observed.

TABLE 18 DoE2 screening: pH (light exposure) Salt Surfactant (NaCl)Buffer (Polysorbate 80) Form concentration type concentration Time After# (mM) (10 mM) pH Stabilizer (mg/mL) 0 exposure DoE2-1 50 Acetate 5.2Trehalose 0 5.2 5.2 dihydrate (200 mM) DoE2-2 50 Acetate 5.2 Trehalose0.5 5.2 5.3 dihydrate (200 mM) DoE2-3 50 Acetate 5.2 Trehalose 1 5.2 5.3dihydrate (200 mM)2.17 Effect of Surfactant on Freeze-Thawing Cycles

Isoforms profiles, aggregates and sub-visible particles of the threeDoE2 formulations have been determined before and after fivefreeze-thawing cycles (−80° C.→room temperature) in order to assesswhether the surfactant exerts any impact.

FIG. 18 is a bar chart showing the main peak isoforms profile, asdetermined by iCE280 analysis, of the DoE2 formulations (of Example 2)before (blue bars, time=0) and after m² (red bars) five freeze-thawingcycles (−80° C.→room temperature).

FIG. 19 is a bar chart showing the acid cluster peak(s) isoformsprofile, as determined by iCE280 analysis, of the DoE2 formulations (ofExample 2) before (blue bars, time=0) and after m² (red bars) fivefreeze-thawing cycles (−80° C.→room temperature).

FIG. 20 is a bar chart showing the % aggregation, as determined bySE-HPLC, of the DoE2 formulations (of Example 2), along with referencestandards (representing comparator HUMIRA® formulations) before (bluebars, time=0) and after m² (red bars) five freeze-thawing cycles (−80°C.→room temperature).

FIG. 21 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 10microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

FIG. 22 is a bar chart showing the number concentration (#/mg) ofsub-visible particles with a particle size less than or equal to 25microns, as determined by sub-visible particle count analysis, of theDoE2 formulations (of Example 2) before (blue bars, time=0) and after m²(red bars) five freeze-thawing cycles (−80° C.→room temperature).

No changes in isoforms and in aggregates were observed (FIGS. 18-20)upon freeze-thawing, whilst minimal, non-critical changes (FIGS. 21-22)in sub-visible particles were highlighted, and found to be non-relatedto the presence of surfactant.

Conclusion of Screening Experiment 2

On the basis of the data collected, relevant to thermal, mechanical andlight stress, the following conclusion can be made:

Formulations in 10 mM Sodium Acetate/Acetic Acid Buffer at pH 5.2(DoE2-1, DoE2-2, DoE2-3):

-   -   Upon thermal stress, performances comparable or better (lower        increase in aggregation) than Humira were highlighted.        Beneficial impact deriving from higher Polysorbate 80        concentrations.    -   No change in the quality attributes upon mechanical shaking.    -   Improved performances with respect to Humira when subjected to        continuous (7 hours) irradiation.

Based on the screening work carried out on different formulationsvarying in buffer/pH, stabilizer, isotonicity agent (NaCl) amount andsurfactant (Polysorbate 80) level, the best composition, showingcomparable or even improved characteristics with respect to Humira upondifferent stressing conditions (thermal, mechanical, light) has beenidentified as:

Ingredient Amount (mg/mL) Adalimumab 50    Acetic Acid glacial 100%  0.60 * Trehalose dihydrate   75.67 ** Polysorbate 80 1.00 Sodiumchloride    2.92 *** WFI and sodium hydroxide q.b. to adjust pH to 5.2 *corresponding to 10 mM sodium acetate/acetic acid buffer; **corresponding to 200 mM; *** corresponding to 50 mM

Such formulations can be readily incorporated within pre-filled glasssyringes with 29 G ½″ needles).

Abbreviations

-   DoE Design of experiment-   DP Drug product-   DS Drug substance-   DSF Differential scanning fluorimetry-   OD Optical density-   PES Polyethersulphone-   rpm rounds per minute-   RT Room Temperature-   SE-HPLC Size exclusion high performance liquid chromatography-   SMI Summary manufacturing instructions-   SOP Standard operating procedure-   WI Working instruction    Clauses    1. A liquid pharmaceutical composition comprising:    -   (a) adalimumab;    -   (b) an acetate buffering agent (or acetate buffer system); and    -   (c) a sugar stabiliser selected from the group including        trehalose, mannitol, sucrose, maltose, lactose, xylitol,        arabitol, erythritol, lactitol, maltitol, and inositol; wherein        the composition is either (substantially or entirely) free of        arginine or comprises arginine in a concentration of at most 0.1        mM.        2. The liquid pharmaceutical composition according to clause 1,        wherein the composition further comprises an acid/base conjugate        of the buffering agent such that together the buffering agent        and its acid/base conjugate are present at a level and in a        relative amount sufficient for the composition to have a pH        between pH 5.0 and 5.5, wherein the acetate buffering agent is        sodium acetate and the acid/base conjugate is acetic acid.        3. The liquid pharmaceutical composition according to any        preceding clause, wherein the sugar stabiliser is trehalose.        4. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition is either        (substantially or entirely) free of amino acids or comprises one        or more amino acids in a (collective) concentration of at most        0.1 mM.        5. The liquid pharmaceutical composition according to clause 4,        wherein the composition is free of amino acids.        6. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition is either        (substantially or entirely) free of surfactants, with the        optional exception of polysorbate 80, or comprises one or more        of surfactants (optionally excluding polysorbate 80) in a        (collective) concentration of at most 0.001 mM.        7. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition is (substantially or        entirely) free of polysorbate 20.        8. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition is either        (substantially or entirely) free of phosphate buffering agents        or comprises a phosphate buffer system in a concentration of at        most 0.1 mM.        9. The liquid pharmaceutical composition according to any        preceding clause, further comprising a tonicifier selected from        sodium chloride, potassium chloride, magnesium chloride, or        calcium chloride.        10. The liquid pharmaceutical composition according to any        preceding clause, further comprising polysorbate 80.        11. The liquid pharmaceutical composition according to any        preceding clause, wherein the osmolality of the composition is        between 220 and 400 mOsm/kg.        12. The liquid pharmaceutical composition according to any        preceding clause, wherein the protein unfolding temperature of        adalimumab in the liquid pharmaceutical composition is greater        than or equal to 70° C.        13. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition comprises adalimumab,        acetate buffering species, and trehalose in a weight ratio of        25-75:0.12-3.0:15-140 respectively.        14. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition comprises adalimumab,        acetate buffering species, trehalose, and sodium chloride in a        weight ratio of 45-55:0.30-0.84:65-72:2.7-3.1 respectively.        15. The liquid pharmaceutical composition according to any        preceding clause, wherein the composition comprises:    -   45 to about 55 mg/ml adalimumab;    -   5 to 14 mM sodium acetate/acetic acid buffer system;    -   190 to 210 mM trehalose;    -   40 to 60 mM sodium chloride;    -   0.9 mg/mL and 1.1 mg/mL polysorbate 80; and    -   water (for injection);    -   wherein the composition:        -   has a pH between 5.1 and 5.3;        -   is free of arginine or comprises arginine in a concentration            of at most 0.001 mM;        -   is free of amino acids or comprises one or more amino acids            in a (collective) concentration of at most 0.001 mM.        -   is free of surfactants with the exception of polysorbate 80            or comprises one or more of said surfactants (excluding            polysorbate 80) in a (collective) concentration of at most            0.0001 mM; and/or        -   is free of phosphate buffering agents (e.g. sodium            dihydrogen phosphate, disodium hydrogen phosphate) or            comprises a phosphate buffer system in a concentration of at            most 0.001 mM.            16. A drug delivery device comprising a liquid            pharmaceutical composition according to any preceding            clause.            17. A liquid pharmaceutical composition according to any of            clauses 1 to 15 for use in the treatment of rheumatoid            arthritis, psoriatic arthritis, ankylosing spondylitis,            Crohn's disease, ulcerative colitis, moderate to severe            chronic psoriasis and/or juvenile idiopathic arthritis.

The invention claimed is:
 1. An aqueous pharmaceutical composition,wherein the composition comprises: (a) 45 to 55 mg/mL adalimumab; (b) 5to 50 mM acetate buffer system; (c) 150 to 250 mM sugar stabiliser,wherein the sugar stabiliser is trehalose; and (d) 0.9 to 1.1 mg/mLpolysorbate 80; and wherein the composition: has a pH between 5.1 and5.3; is free of arginine; is free of phosphate buffering agents; is freeof amino acids; is free of surfactants, with the exception ofpolysorbate 80; and comprises at most one sugar stabilizer, and at mostone buffer system.
 2. The aqueous pharmaceutical composition of claim 1,wherein the composition comprises only one non-buffering tonicifierwhich is the sugar stabilizer.
 3. The aqueous pharmaceutical compositionof claim 1, wherein the composition comprises the adalimumab at aconcentration of 50 mg/mL.
 4. The aqueous pharmaceutical composition ofclaim 3, wherein the composition consists of: (a) 50 mg/mL adalimumab;(b) 10-50 mM acetate buffer system; (c) 190-250 mM trehalose; (d) 1mg/mL polysorbate 80; and (e) water; wherein the composition has a pH ofpH 5.2.
 5. A drug delivery device comprising the aqueous pharmaceuticalcomposition of claim
 1. 6. A drug delivery device comprising the aqueouspharmaceutical composition of claim
 2. 7. A drug delivery devicecomprising the aqueous pharmaceutical composition of claim
 3. 8. A drugdelivery device comprising the aqueous pharmaceutical composition ofclaim
 4. 9. A method of treating rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis,moderate to severe chronic psoriasis, or juvenile idiopathic arthritisin a patient in need of such treatment, said method comprisingadministering to said patient a therapeutically effective amount of theaqueous pharmaceutical composition of claim
 1. 10. A method of treatingrheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,Crohn's disease, ulcerative colitis, moderate to severe chronicpsoriasis, or juvenile idiopathic arthritis in a patient in need of suchtreatment, said method comprising administering to said patient atherapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 2. 11. A method of treating rheumatoid arthritis,psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerativecolitis, moderate to severe chronic psoriasis, or juvenile idiopathicarthritis in a patient in need of such treatment, said method comprisingadministering to said patient a therapeutically effective amount of theaqueous pharmaceutical composition of claim
 3. 12. A method of treatingrheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis,Crohn's disease, ulcerative colitis, moderate to severe chronicpsoriasis, or juvenile idiopathic arthritis in a patient in need of suchtreatment, said method comprising administering to said patient atherapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 4. 13. A method of treating psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis, or juvenile idiopathic arthritis in a patientin need of such treatment, said method comprising administering to saidpatient a therapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 1. 14. A method of treating psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis, or juvenile idiopathic arthritis in a patientin need of such treatment, said method comprising administering to saidpatient a therapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 2. 15. A method of treating psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis, or juvenile idiopathic arthritis in a patientin need of such treatment, said method comprising administering to saidpatient a therapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 3. 16. A method of treating psoriatic arthritis,ankylosing spondylitis, Crohn's disease, ulcerative colitis, moderate tosevere chronic psoriasis, or juvenile idiopathic arthritis in a patientin need of such treatment, said method comprising administering to saidpatient a therapeutically effective amount of the aqueous pharmaceuticalcomposition of claim
 4. 17. The aqueous pharmaceutical composition ofclaim 1, where the composition consists of: (a) 45 to 55 mg/mLadalimumab; (b) a single buffer system, which is an acetate buffersystem in a molar ratio of acetate buffer system to adalimumab of from5:1 to 145:1; (c) trehalose at a molar ratio of trehalose to adalimumabof from 430:1 to 720:1; (d) polysorbate 80 at a molar ratio ofpolysorbate 80 to adalimumab of from 2.1:1 to 2.3:1; and (e) water;wherein the composition has a pH between 5.1 and 5.3.